Peptides and combination of peptides for use in immunotherapy against leukemias and other cancers

ABSTRACT

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/949,884, filed Apr. 10, 2018, which claims priority to U.S.Provisional Application 62/483,690, filed Apr. 10, 2017, and GermanPatent Application 10 2017 107 710.3, filed on Apr. 10, 2017. Each ofthese applications is incorporated by reference in its entirety.

This application also is related to PCT/EP2018/059114, filed Apr. 10,2018, the content of which is incorporated herein by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “2912919-086003_Sequence_Listing_ST25.txt” createdon 20 Feb. 2020, and 43,507 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T-cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules.

The present invention relates to several novel peptide sequences andtheir variants derived from HLA class I molecules of human tumor cellsthat can be used in vaccine compositions for eliciting anti-tumor immuneresponses, or as targets for the development ofpharmaceutically/immunologically active compounds and cells.

BACKGROUND OF THE INVENTION

According to the World Health Organization (WHO), cancer ranged amongthe four major non-communicable deadly diseases worldwide in 2012. Forthe same year, colorectal cancer, breast cancer and respiratory tractcancers were listed within the top 10 causes of death in high incomecountries (http://www.who.int/mediacentre/factsheets /fs310/en/).

Epidemiology

In 2012, 14.1 million new cancer cases, 32.6 million patients sufferingfrom cancer (within 5 years of diagnosis) and 8.2 million cancer deathswere estimated worldwide (Ferlay et al., 2013; Bray et al., 2013).

Within the group of leukemia, the current invention specifically focuseson chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML)and acute myeloid leukemia (AML).

CLL is the most common leukemia in the Western world where it comprisesabout one third of all leukemia. Incidence rates are similar in the USand Europe, and estimated new cases are about 16,000 per year. CLL ismore common in Caucasians than in Africans, rarer in Hispanics andNative Americans and seldom in Asians. In people of Asian origin, CLLincidence rates are 3-fold lower than in Caucasians (Gunawardana et al.,2008). The five-year overall survival for patients with CLL is about79%.

AML is the second most common type of leukemia diagnosed in both adultsand children. Estimated new cases in the United States are about 21,000per year. The five-year survival rate of people with AML isapproximately 25%.

CML accounts for 15-25% of adult leukemias with incidences ranging at1.2/100,000 in Europe and 1.75/100,000 in the US (Hoglund et al., 2015).The number of estimated new CML cases in the US are 8,220 for the year2016 with an estimated number of 1,070 deaths (National CancerInstitute, 2015). Since the introduction of imatinib in the year 2000,annual mortality in CML has decreased from 10-20% down to 1-2%. As aconsequence, prevalence of CML in the US has increased from an estimated25,000-30,000 cases in 2000 to 80,000-100,000 in 2015 (Huang et al.,2012).

Therapy

Chronic lymphocytic leukemia—While CLL is not curable at present, manypatients show only slow progression of the disease or worsening ofsymptoms. As patients do not benefit from an early onset of treatment,the initial approach is “watch and wait” (Richards et al., 1999). Forpatients with symptomatic or rapidly progressing disease, severaltreatment options are available. These include chemotherapy, targetedtherapy, immune-based therapies like monoclonal antibodies, chimericantigen-receptors (CARs) and active immunotherapy, and stem celltransplants.

Chemotherapeutic drugs used for CLL treatment are mostly alkylatingagents like chlorambucil and cyclophosphamide or purine analogues likefludarabine. The German CLL Study Group (GCLLSG) CCL4 demonstrated thatfludarabine/cyclophosphamide combinational therapy is superior to solefludarabine treatment (complete remission (CR) of 24% vs.7%) (Eichhorstet al., 2006).

Ibrutinib and idelalisib are kinase inhibitors that target molecules inthe B-cell receptor signaling cascade. Ibrutinib inhibits Bruton'styrosine kinase (BTK), a src-related cytoplasmic tyrosine kinaseimportant for B-cell maturation, and is used for initial or second-linetherapy (Byrd et al., 2013; O'Brien et al., 2014). Idelalisib is aPI3K-delta inhibitor used in combination with rituximab in refractoryCLL (Furman et al., 2014).

Hematopoietic stem cell transplants (HSCTs) can be considered forpatients with poor prognosis, e.g. patients with deletions at chromosome17p (del 17p) or p53 mutations. HSCTs can either be allogeneic, wherethe transplanted cells are donated from an HLA-matched person, orautologous, where the patients' own stem cells are re-infused afterchemotherapy (Schetelig et al., 2008).

Monoclonal antibodies are widely used in hematologic malignancies. Thisis due to the knowledge of suitable antigens based on the goodcharacterization of immune cell surface molecules and the accessibilityof tumor cells in blood or bone marrow. Common monoclonal antibodiesused in CLL therapy target either CD20 or CD52. Rituximab, the firstmonoclonal anti-CD20 antibody originally approved by the FDA fortreatment of NHLs, is now widely used in CLL therapy. Combinationaltreatment with rituximab/fludarabine/cyclophosphamide leads to higher CRrates and improved overall survival (OS) compared to the combinationfludarabine/cyclophosphamide and has become the preferred treatmentoption (Hallek et al., 2008). Ofatumomab targets CD20 and is used fortherapy of refractory CLL patients (Wierda et al., 2011). Obinutuzumabis another monoclonal anti-CD20 antibody used in first-line treatment incombination with chlorambucil (Goede et al., 2014).

Alemtuzumab is an anti-CD52 antibody used for treatment of patients withchemotherapy-resistant disease or patients with poor prognostic factorsas del 17p or p53 mutations (Parikh et al., 2011).

Novel monoclonal antibodies target CD37 (otlertuzumab, BI 836826,IMGN529 and (177)Lu-tetulomab) or CD40 (dacetuzumab and lucatumumab) andare tested in pre-clinical settings (Robak and Robak, 2014).

Several completed and ongoing trials are based on engineered autologouschimeric antigen receptor (CAR)-modified T cells with CD19 specificity(Maus et al., 2014). So far, only the minority of patients showeddetectable or persistent CARs. One partial response (PR) and twocomplete responses (CR) have been detected in the CAR T-cell trials byPorter et al. and Kalos et al. (Kalos et al., 2011; Porter et al.,2011).

Active immunotherapy includes the following strategies: gene therapy,whole modified tumor cell vaccines, DC-based vaccines and tumorassociated antigen (TAA)-derived peptide vaccines.

Approaches in gene therapy make use of autologous genetically modifiedtumor cells. These B-CLL cells are transfected withimmuno-(co-)stimulatory genes like IL-2, IL-12, TNF-alpha, GM-CSF, CD80,CD4OL, LFA-3 and ICAM-1 to improve antigen presentation and T cellactivation (Carballido et al., 2012). While specific T-cell responsesand reduction in tumor cells are readily observed, immune responses areonly transient.

Several studies have used autologous DCs as antigen presenting cells toelicit anti-tumor responses. DCs have been loaded ex vivo with tumorassociated peptides, whole tumor cell lysate and tumor-derived RNA orDNA. Another strategy uses whole tumor cells for fusion with DCs andgeneration of DC-B-CLL-cell hybrids. Transfected DCs initiated both CD4+and CD8+ T-cell responses (Muller et al., 2004). Fusion hybrids and DCsloaded with tumor cell lysate or apoptotic bodies increasedtumor-specific CD8+ T-cell responses. Patients that showed a clinicalresponse had increased IL-12 serum levels and reduced numbers of Tregs(Palma et al., 2008).

Different approaches use altered tumor cells to initiate or increaseCLL-specific immune responses. An example for this strategy is thegeneration of trioma cells: B-CLL cells are fused to anti-Fc receptorexpressing hybridoma cells that have anti-APC specificity. Trioma cellsinduced CLL-specific T-cell responses in vitro (Kronenberger et al.,2008).

Another strategy makes use of irradiated autologous CLL cells withBacillus Calmette-Guerin as an adjuvant as a vaccine. Several patientsshowed a reduction in leukocyte levels or stable disease (Hus et al.,2008).

Besides isolated CLL cells, whole blood from CLL patients has been usedas a vaccine after preparation in a blood treatment unit. The vaccineelicited CLL-specific T-cell responses and led to partial clinicalresponses or stable disease in several patients (Spaner et al., 2005).

Several TAAs are over-expressed in CLL and are suitable forvaccinations. These include fibromodulin (Mayr et al., 2005),RHAMM/CD168 (Giannopoulos et al., 2006), MDM2 (Mayr et al., 2006), hTERT(Counter et al., 1995), the oncofetal antigen-immature laminin receptorprotein(OFAiLRP) (Siegel et al., 2003), adipophilin (Schmidt et al.,2004), survivin (Granziero et al., 2001), KW1 to KW14 (Krackhardt etal., 2002) and the tumor-derived IgVHCDR3 region (Harig et al., 2001;Carballido et al., 2012). A phase I clinical trial was conducted usingthe RHAMM-derived R3 peptide as a vaccine. 5 of 6 patients haddetectable R3-specific CD8+ T-cell responses (Giannopoulos et al.,2010).

Chronic myeloid leukemia—CML is a myeloproliferative neoplasmcharacterized by the (9;22)(q34;q11.2) chromosomal translocationresulting in BCR-ABL1 gene fusion. The resultant BCR-ABL1 fusion proteinshows dysregulated tyrosine kinase activity and plays a central role inthe pathogenesis and maintenance of CML (Lugo et al., 1990). Thediscovery of this molecular mechanism of CML leukemogenesis led to thedevelopment and successful clinical application of the BCR-ABL1 specifictyrosine kinase inhibitors (TKI), which was spearheaded by approval ofimatinib for the first-line treatment of newly diagnosed CML in 2002(Johnson et al., 2003). The introduction of TKIs drastically alteredpatient management and clinical outcome in CML and led to a vastlyimproved life expectancy of CML patients (Schmidt, 2016). However, TKItherapy has to be maintained lifelong which increases the risk ofdeveloping secondary resistance (Khorashad et al., 2013) and comes atsignificant cost (Kantarjian et al., 2013). Furthermore, albeitgenerally well tolerated, distinct toxicity profiles may prohibitapplication of certain TKI in patients with comorbidities and may leadto rare but severe adverse events (Jabbour and Kantarjian, 2016).Currently, the only curative therapy for CML is allogeneic stem celltransplantation, which due to significant morbidity and mortality isconfined to patients diagnosed in advanced phase or used as a salvageoption for patients that failed multiple TKI (Horowitz et al., 1996;Radich, 2010).

CML is classified into three clinical phases of chronic phase,accelerated phase and blast crisis based on the frequency of CML blastsin blood and bone marrow. Around 90% of patients are diagnosed in theinitial chronic phase, with around 50% of all newly diagnosed patientsbeing asymptomatic (Jabbour and Kantarjian, 2016). Before theintroduction of TKI, CML drug therapy was limited to unspecific agentssuch as combination chemotherapy with the alkylating agent busulfan andthe ribonucleotide reductase inhibitor hydroxyurea or treatment withinterferon-alfa (IFN-a). In these settings, the chronic phase had amedian duration of 3 to 5 years followed by an accelerated phase of 3-6months, which generally terminated fatally (Silver et al., 1999).Although IFN-a induced disease regression and improved survival in asubset of patients it was hindered by significant toxicity and lack ofefficacy (Kujawski and Talpaz, 2007) and was ultimately replaced by TKItherapy. The advent of targeted therapy for CML using TKI drasticallyaltered the course of disease and increased the 10-year survival ratefrom approximately 20% to 80-90% (Jabbour and Kantarjian, 2016).

Currently five TKIs are approved in the US for the treatment of CMLpatients. The standard first-line therapy of chronic phase CML usuallyconsists treatment with either the first-generation TKI Imatinib(O′Brien et al., 2003) or one of the second generation TKIs nilotinib(Saglio et al., 2010) or dasatinib (Kantarjian et al., 2010). Bosutinib(Cortes et al., 2012) and ponatinib are indicated for patients withintolerance or resistance to prior TKI.

Despite high response rates and deep responses, primary and secondaryresistance to TKI therapy has been observed in CML patients. The bestcharacterized resistance mechanisms are genomic amplification ofBCR-ABL1 or mutations to the BCR-ABL1 kinase domain (Khorashad et al.,2013). Mutation analysis is typically performed after progression underTKI therapy and may guide the selection of subsequent TKIs (Soverini etal., 2011). For patients showing resistance or intolerance to two ormore TKIs, the unspecific protein translation inhibitor omacetaxine isapproved in the US (Gandhi et al., 2014), although TKI are generally thepreferred option (Soverini et al., 2011). Omacetaxine is a BCR-ABL1independent drug which has been shown to induce apoptosis in primary CMLstem cells through downregulation of the anti-apoptotic McI1-1 protein(Allan et al., 2011).

Cessation of imatinib treatment has been investigated in the StopImatinib (STIM) trial, which enrolled patients with complete molecularresponses (CMR) of more than two years (Mahon et al., 2010). In a recentupdate, 61% of patients had experienced a molecular relapse, with 95% ofevents occurring within 7 months of stopping imatinib. Of note, almostall patients remained sensitive to imatinib and achieved CMR onceimatinib was restarted. Similar observations were made in the TWISTERtrial (Ross et al., 2013). However, while these studies demonstrate thatstopping TKI therapy is feasible and some patients may be cured, TKIdiscontinuation still should only be performed under within the confinesof clinical trials and requires rigorous patient monitoring (Jabbour andKantarjian, 2016). The main reason for molecular relapse afterdiscontinuation of TKI therapy is the presence of minimal residualdisease (MRD), i.e. the persistence of residual CML stem cells in thebone marrow (Bhatia et al., 2003). These CML stem cells have been shownto persist independently of BCR-ABL1 activity and thus cannoteffectively be eliminated by TKI therapy (Corbin et al., 2011).

Together, this calls for novel therapeutic strategies targeting theBCR-ABL1 independent pool of CML stem cells to achieve eradication ofMRD. The fact that allogeneic stem cell transplantation and unspecificimmunotherapy with IFN-a can induce long-term remissions in a subset ofpatients indicates that immunological targeting of CML is a viabletherapeutic option. Antigen-specific immunotherapy targeting BCR-ABLjunction peptides has been shown to induce of peptide-specific T cellresponses followed by antitumor effects in a substantial subset ofpatients in two independent clinical phase II vaccination trials(Bocchia et al., 2005; Cathcart et al., 2004). However, targeting ofthese CML-specific targets is confined to a subset of the patientcollective expressing the appropriate HLA allotypes. Other targets ofCML immunotherapy include overexpressed antigens such as the zinc fingertranscription factor WT1, which has been shown to induce cytotoxic CD8 Tcells capable of killing WT1⁺ in clinical vaccination trials of myeloidmalignancies (Rezvani et al., 2008; Keilholz et al., 2009). Furthermore,a T cell receptor-like antibody specific for the WT1-derived HLA-A*02peptide RMFPNAPYL has been shown to mediate antibody-dependent cellularcytotoxicity in human leukemia xenografts (Dao et al., 2013). Furtherleukemia-associated antigens described in CML include the Receptor forhyaluronan acid-mediated motility (RHAMM) (Greiner et al., 2002),PPP2R5C (Zheng et al., 2011), PR1, PR3, PPP2R5C, ELA2, PRAME (Smahel,2011) as well as an epitope derived from the M-phase phosphoprotein 11protein (MPP11) (Al et al., 2010). The antigens described in thesestudies were typically identified using reverse immunology approachesand predictions and lack direct evidence of CML-associated presentationby HLA molecules.

Immunostimulatory treatment of CML is being revisited in the age of TKItherapy as exemplified by studies assessing IFN-a in combination withTKI in patients with the multi-TKI resistant T315I mutation (Itonaga etal., 2012). One case report found induction of a sustained deepmolecular response in a single patient, who could discontinue therapypander et al., 2014). Furthermore, ongoing trials are investigating theefficacy of immune checkpoint inhibitors such as α-CTLA4 (ipilimumab)and α-PD1 (nivolumab). Antigen-specific (combination) therapy thusrepresents a promising avenue for the eradication of MRD in CML as wellas for the treatment of multi-TKI resistant clones.

Acute myeloid leukemia—AML treatment is divided into two phases:induction therapy and post-remission/“consolidation therapy”. Inductiontherapy is administered to induce remission and consists ofcombinational chemotherapy. Consolidation therapy consists of additionalchemotherapy or hematopoietic cell transplantation (HCT) (Showel andLevis, 2014).

The most common chemotherapeutic drugs used to treat AML are cytarabine,daunorubicin, idarubicin and mitoxantrone followed by cladribine,fludarabine and diverse others. Azacytidine and decitabine (DNAhypomethylating agents) are now used for treatment of MDS/AML. Treatmentfor APL/AML M3 includes all-trans retinoic acid (ATRA) and arsenictrioxide (ATO) (National Cancer Institute, 2015).

“Standard treatment” for AML is considered as “3+7”: 3 days ofidarubicin or daunorubicin and 7 days of cytarabine, followed by severalsimilar courses to achieve complete remission (CR) (Estey, 2014). Thedecision between standard therapy and clinical trial is based on therisk stratification.

AML cases with intermediate-risk karyotype show either no karyotypicabnormalities or only one or two abnormalities not categorized as high-or low-risk.

FLT3 mutations are associated with an aggressive type of AML and a poorprognosis. They often occur together with NPM1 and DNMT3a (DNAmethyltransferase 3A) mutations. NPM1 (nucleophosmin) mutations are afavorable prognostic indicator, if not found together with FLT3mutations. CEPBA (CCAAT-enhancer-binding protein alpha/C/EBPa) mutationsconfer a survival advantage in the case of double or homozygous CEBPAmutations without wild-type expression. Altered methylation patterns ina variety of genes are caused by mutations in isocitrate dehydrogenase(IDH1 and IDH2) and DNMT3A. These mutations are associated with poorsurvival.

AML cases with favorable-risk karyotype consist of APL (acutepromyelocytic leukemia) and CBF (core-binding factor) leukemias. APLcases are associated with the fusion of the myeloid transcription factorPML to the retinoic acid receptor subunit alpha (RARA). The PML/RARAtranslocation is a favorable prognostic mutation. CBF leukemia casesshow translocations involving a subunit of CBF. In t(8;21) CBF alpha isfused to the ETO gene. In inv(16) CBF beta is fused to the smooth musclemyosin heavy chain. CBF translocations are very favorable prognosticmutations.

AML cases with unfavorable-risk karyotype are characterized by a complexkaryotype including chromosomal aberrations such as translocations,unbalanced rearrangements and gains/losses of whole chromosomes. Theyare associated with a poor prognosis.

MDS/AML cases evolve from myelodysplastic syndromes and carry a worseprognosis than other AML sub-groups (Showel and Levis, 2014).

Besides the above-listed prognostic factors, additional molecularmarkers or marker combinations can be used to judge the prognosis inspecific cytogenetic subsets:

TP53 mutations are the most unfavorable genetic alteration in AML. NPM1mutated and FLT3 WT together with a mutation in IDH1 or IDH2 is seen asfavorable. Unfavorable factors include a partial tandem duplication inthe MLL gene, a mutated TET2 gene, FLT3 ITD +together with a mutation inDNMT3a and CEBPA, FLT3 ITD—together with a mutation in ASXL1 or PHF6,and CD25 expression (stem cell-like “signature” and poorer outcome). Thepresence of CKIT mutations converts the prognosis of patients with afavorable inv(16) or t(8;21) into a more intermediate range. SPARC isup-regulated in NK (normal karyotype) patients with unfavorable geneexpression signature and down-regulated in association with thefavorable NPM1 mutation. miR-155 over-expression conveys a poorprognosis in NK AML. Differentially methylated regions (DMRs) areprognostic when found in association with several genes (FLT3 mutation,NPM1 mutation). In this case, a lower expression is associated with abetter prognosis (Estey, 2014).

Post-treatment information/ information about minimal residual disease(MRD) should be included into following treatment decisions. Theseinclude the response to induction therapy, PCR of fusion transcripts,mutated genes and over-expressed genes to detect MRD and multi-parameterflow cytometry for observation of aberrant expression of surfaceantigens.

Clinical trials are recommended for patients who belong to theprognostic groups unfavorable and intermediate-2. Treatment optionsinclude hypomethylating agents (HMAs) as Azacytidine or decitabine,CPX-351, which is a liposomal formulation of daunorubicin and cytarabinein a 1:5 “optimal” molar ratio, and volasertib, which is an inhibitor ofpolo kinases. Volasertib is given in combination with LDAC (low-dosecytarabine). Several different FLT3 inhibitors can be administered incase of FLT3 mutations. These include sorafenib, which is given incombination with 3+7, quizartinib, a more selective inhibitor of FLT3ITD that also inhibits CKIT, crenolanib, and midostaurin, an unselectiveFLT3 ITD inhibitor. Another treatment option is targeting CD33 withantibody-drug conjugates (anti-CD33 + calechiamicin, SGN-CD33a,anti-CD33+ actinium-225), bispecific antibodies (recognition of CD33 +CD3 (AMG 330) or CD33+ CD16) and chimeric antigen receptors (CARs)(Estey, 2014).

Considering the severe side-effects and expense associated with treatingcancer, there is a need to identify factors that can be used in thetreatment of cancer in general and chronic lymphocytic leukemia, chronicmyeloid leukemia and acute myeloid leukemia in particular. There is alsoa need to identify factors representing biomarkers for cancer in generaland chronic lymphocytic leukemia, chronic myeloid leukemia and acutemyeloid leukemia in particular, leading to better diagnosis of cancer,assessment of prognosis, and prediction of treatment success.

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens.

The current classification of tumor associated antigens (TAAs) comprisesthe following major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members and NY-ESO-1.

b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Many of these melanocyte lineage-related proteins are involved inbiosynthesis of melanin and are therefore not tumor specific butnevertheless are widely used for cancer immunotherapy. Examples include,but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma orPSA for prostate cancer.

c) Over-expressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir over-expression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.

d) Tumor-specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor-specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor-(-associated) exon in case of proteins with tumor-specific(-associated) isoforms.

e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.

f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, defective ribosomal products (DRIPs) and largerpeptides. However, peptides derived from endosomal compartments orexogenous sources are also frequently found on MHC class I molecules.This non-classical way of class I presentation is referred to ascross-presentation in the literature (Brossart and Bevan, 1997; Rock etal., 1990). MHC class II molecules can be found predominantly onprofessional antigen presenting cells (APCs), and primarily presentpeptides of exogenous or transmembrane proteins that are taken up byAPCs e.g. during endocytosis, and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positive Tcells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of immense importance for the developmentof pharmaceutical products for triggering anti-tumor immune responses(Gnjatic et al., 2003). At the tumor site, T helper cells, support acytotoxic T cell-(CTL-) friendly cytokine milieu (Mortara et al., 2006)and attract effector cells, e.g. CTLs, natural killer (NK) cells,macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al.,2006).

Longer (elongated) peptides of the invention can act as MHC class IIactive epitopes.

T-helper cells, activated by MHC class II epitopes, play an importantrole in orchestrating the effector function of CTLs in anti-tumorimmunity. T-helper cell epitopes that trigger a T-helper cell responseof the TH1 type support effector functions of CD8-positive killer Tcells, which include cytotoxic functions directed against tumor cellsdisplaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFNy) (Beatty andPaterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T cellsas direct anti-tumor effectors (Braumuller et al., 2013; Tran et al.,2014).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T cells (ligand: MHC class I molecule+peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-class-I-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T cells bearingspecific T cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. In a preferred embodiment, the peptide should beover-presented by tumor cells as compared to normal healthy tissues. Itis furthermore desirable that the respective antigen is not only presentin a type of tumor, but also in high concentrations (i.e. copy numbersof the respective peptide per cell). Tumor-specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g. in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated and thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004). It isessential that epitopes are present in the amino acid sequence of theantigen, in order to ensure that such a peptide (“immunogenic peptide”),being derived from a tumor associated antigen, leads to an in vitro orin vivo T-cell-response.

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a T cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are usually based on the useof T-cells that can be isolated from patients or healthy subjects, orthey are based on the generation of differential transcription profilesor differential peptide expression patterns between tumors and normaltissues. However, the identification of genes over-expressed in tumortissues or human tumor cell lines, or selectively expressed in suchtissues or cell lines, does not provide precise information as to theuse of the antigens being transcribed from these genes in an immunetherapy. This is because only an individual subpopulation of epitopes ofthese antigens are suitable for such an application since a T cell witha corresponding TCR has to be present and the immunological tolerancefor this particular epitope needs to be absent or minimal. In a verypreferred embodiment of the invention it is therefore important toselect only those over- or selectively presented peptides against whicha functional and/or a proliferating T cell can be found. Such afunctional T cell is defined as a T cell, which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs)and antibodies or other binding molecules (scaffolds) according to theinvention, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 279 or a variant sequencethereof which is at least 77%, preferably at least 88%, homologous(preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 toSEQ ID NO: 279, wherein said variant binds to MHC and/or induces T cellscross-reacting with said peptide, or a pharmaceutical acceptable saltthereof, wherein said peptide is not the underlying full-lengthpolypeptide.

The present invention further relates to a peptide of the presentinvention comprising a sequence that is selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 279 or a variant thereof, whichis at least 77%, preferably at least 88%, homologous (preferably atleast 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 279,wherein said peptide or variant thereof has an overall length of between8 and 100, preferably between 8 and 30, and most preferred of between 8and 14 amino acids.

The following tables show the peptides according to the presentinvention, their respective SEQ ID NOs, and the prospective source(underlying) genes for these peptides. In Table 1, peptides with SEQ IDNO: 1 to SEQ ID NO: 16 bind to HLA-A*01, peptides with SEQ ID NO: 17 toSEQ ID NO: 27 bind to HLA-A*02, peptides with SEQ ID NO: 28 to SEQ IDNO: 52 bind to HLA-A*03, peptides with SEQ ID NO: 53 to SEQ ID NO: 76bind to HLA-A*24, peptides with SEQ ID NO: 77 to SEQ ID NO: 106 bind toHLA-B*07, peptides with SEQ ID NO: 107 to SEQ ID NO: 121 bind toHLA-B*08, peptides with SEQ ID NO: 122 to SEQ ID NO: 187 bind toHLA-B*44. The peptides in Table 2 have been disclosed before in largelistings as results of high-throughput screenings with high error ratesor calculated using algorithms, but have not been associated with cancerat all before. In Table 2, peptides with SEQ ID NO: 188 to SEQ ID NO:196 bind to HLA-A*01, peptides with SEQ ID NO: 197 to SEQ ID NO: 207bind to HLA-A*02, peptides with SEQ ID NO: 208 to SEQ ID NO: 221 bind toHLA-A*03, peptides with SEQ ID NO: 222 to SEQ ID NO: 224 bind toHLA-A*24, peptides with SEQ ID NO: 225 to SEQ ID NO: 255 bind toHLA-B*07, peptide with SEQ ID NO: 256 binds to HLA-B*08, peptides withSEQ ID NO: 257 to SEQ ID NO: 277 bind to HLA-B*44. The peptides in Table3 are additional peptides that may be useful in particular incombination with the other peptides of the invention. In Table 3,peptide with SEQ ID NO: 278 binds to HLA-A*02, peptide with SEQ ID NO:279 binds to HLA-A*24.

TABLE 1 Peptides according to the present invention. Seq Official GeneHLA ID No Sequence Symbol(s) allotype   1 LTEGHSGNYY FCRL5 A*01   2TMIRIFHRY S100Z A*01/B*15   3 YINPAKLTPY CARD11 A*01/A*03   4 ALDQNKMHYCPA3 A*01   5 GTDVLSTRY CPA3 A*01   6 VTEGVAQTSFY HLA-DOA A*01   7FMDSESFYY INPP5F A*01   8 STDSAGSSY PAX5 A*01   9 YSHPQYSSY PAX5A*01/B*15  10 YSDIGHLL SESN3 A*01  11 AAADHHSLY S0X4 A*01/A*32  12ATDIVDSQY T A*01  13 ITDIHIKY VPS13C A*01  14 TFDLTVVSY VPS13C A*01  15SVADIRNAY WDFY4 A*01/A*32  16 WIGDKSFEY ZNF121 A*01/A*29  17 KAYNRVIFVMARCH1 A*02  18 YLLPSVVLL AGPAT5 A*02  19 SLFEGIYTI FLT3 A*02  20FSLEDLVRI KIAA0226L A*02  21 FLFDKLLLI PLEKHG1 A*02  22 ILHAQTLKIRALGPS2 A*02/B*13  23 FAFSGVLRA RREB1 A*02  24 KLGPVAVSI SIPA1L3A*02/B*13  25 YLNEKSLQL ST8SIA6 A*02  26 SLYVQQLKI SYNE2 A*02/B*13  27RLIAKEMNI WDFY4 A*02/B*13  28 VILESIFLK BTK A*03/A*11  29 RIYDEILQSKCD84 A*03  30 RTYGFVLTF DENND5B A*03/A*32  31 ATFNKLVSY DNMT3B A*03/A*32 32 KTSNIVKIK FCRL2 A*03  33 SVFEGDSIVLK FCRL2 A*03/A*11  34 SVYSETSNMDKGSAP A*03/A*11  35 ATKSPAKPK HIST1H1B A*03  36 KAKAAAKPK HIST1H1B A*03 37 KAKKPAGAAK HIST1H1E A*03  38 KARKSAGAAK HIST1H1E A*03  39 IVIQLRAQKHLA-DOB A*03  40 RSKEYIRKK KBTBD8 A*03  41 SVAHLLSKY MAP3K1 A*03/B*15 42 SVSSSTHFTR MAP3K1 A*03/A*11  43 KLMETSMGF MGA A*03/A*32  44KVYDPVSEY MTMR1 A*03/B*15  45 VVFPFPVNK MYCN A*03  46 RVFPSPMRI PLCL2A*03  47 SVLDLSVHK PRDM2 A*03/A*11  48 RIKPPGPTAVPK SCML2 A*03  49GLLEEALFY SMYD3 A*03/A*29  50 GVFNTLISY TARBP1 A*03  51 ASTTVLALK WDFY4A*03/A*11  52 KAFNQSSTLTK ZNF431, ZNF714, A*03 ZNF92, ZNF93  53KYIEYYLVL ADAM28 A*24  54 QQALNFTRF AKAP13 A*24/B*15  55 IFVARLYYFAPOBEC3G A*24  56 KYSSGFRNI ATM A*24  57 RFPPTPPLF BCL11A A*24  58KYLADLPTL CEP85L A*24  59 GLYEGTGRLF DNMT3A, DNMT3B A*24  60 TQDPHVNAFFDOCK10 A*24/B*38  61 IFKEHNFSF FLT3 A*24  62 YYLSHLERI GNA15 A*24  63IYFSNTHFF GPR114 A*24  64 SFQSKATVF HOXA9 A*24  65 AYLKQVLLF INPP5F A*24 66 SQPAVATSF MYB A*24/B*15  67 VFLPSEGFNF N4BP2 A*24  68 LYQDRFDYLNLRP3 A*24  69 EYNTIKDKF PARP15 A*24  70 LYSDIGHLL SESN3 A*24  71RYLGKNWSF SPNS3 A*24  72 TYVENLRLL SYNE2 A*24  73 TYPQLEGFKF WDFY4 A*24 74 SYADNILSF WDR11 A*24  75 RFYLLTEHF ZNF121 A*24  76 KAFSWSSAF ZNF92A*24/A*32  77 RPNGNSLFTSA AFF3 B*07  78 RPRGLALVL CASP2 B*07  79SPVPSHVVMVA CD79B B*07  80 KPLFKVSTF DOCK10 B*07  81 SESPWLHAPSL FAIM3B*07/B*40  82 APFGFLGMQSL FAM129C B*07  83 IPVSRPIL FCRL1 B*07  84SPKLQIAAM FCRL1 B*07  85 IPVSHPVL FCRL3 B*07  86 IPASHPVL FCRL5 B*07  87FPAPILRAV FCRLA B*07  88 MPDPHLYHQM FCRLA B*07  89 FPETVNNLL HEATR5BB*07  90 KPKAAKPKA HIST1H1B B*07  91 KPKAAKPKAA HIST1H1B B*07  92KAKKPAGAA HIST1H1E B*07  93 KARKSAGAA HIST1H1E B*07  94 KPKAAKPKKAAAHIST1H1E B*07  95 KPKAAKPKTA HIST1H1E B*07  96 KPKKAPKSPA HIST1H1E B*07 97 LPFGKIPIL HPGDS B*07/B*51  98 YPIALTRAEM IKZF3 B*07/B*35  99SPRAINNLVL KLHL14 B*07 100 YPYQERVFL PIK3R6 B*07/B*35 101 NPRYPNYMF ROR1B*07 102 LPLSMEAKI RREB1 B*07/B*35 103 IPANTEKASF SCIMP B*07/B*50 104RPMTPTQIGPSL TCL1A B*07 105 NPLTKLLAI TFEC B*07/B*08 106 KAFKWFSALZNF736 B*07 107 QAAQRTAL AFF3 B*08 108 ILAIRQNAL AGPAT5 B*08 109LGHVRYVL AGPAT5 B*08 110 FGLARIYSF CDK6 B*08 111 VTLIKYQEL CLEC17AB*08/A*02 112 APLLRHWEL HLA-DOA B*08/B*07 113 DANSRTSQL MAP3K1 B*08 114HNALRILTF NUP210 B*08 115 ELYQRIYAF PIK3R6 B*08 116 TLKIRAEVL RALGPS2B*08 117 YIKTAKKL RALGPS2 B*08 118 FEKEKKESL SESN3 B*08 119 DLRTKEVVFSIPA1L3 B*08 120 VPPKKHLL TRRAP B*08 121 RPKKVNTL ZBTB24 B*08 122KELPGVKKY ADAM28 B*44 123 EENPGKFLF ARHGAP24 B*44 124 SESLPKEAF ATF7IPB*44 125 SESTFDRTF ATF7IP B*44 126 EENKPGIVY BTLA B*44 127 TEYPVFVYCACNA2D4 B*44 128 GENDRLNHTY CCDC88A B*44 129 GEGAYGKVF CDK6 B*44 130EEEHGKGREY CHD1 B*44 131 EEFETIERF CHD1 B*44 132 GELPAVRDL CIITAB*44/B*40 133 AEHNFVAKA CLEC17A B*44/B*50 134 SEYADTHYF CLNK B*44 135NEIKVYITF CPA3 B*44 136 AEYKGRVTL FAIM3 B*44/B*40/B*49 137 GELGGSVTIFAIM3 B*44/B*49 138 SQAPAARAF FAM129C B*44/B*15 139 RENQVLGSGW FCRL2B*44 140 EYDLKWEF FLT3 B*44/A*23 141 REYEYDLKWEF FLT3 B*44 142 TEIFKEHNFFLT3 B*44 143 YEYDLKWEF FLT3 B*44 144 TEGKRYFTW GANC B*44 145 AEPLVGQRWGDF7 B*44 146 SESKTVVTY ICOSLG B*44 147 KEVPRSYEL IKZF3 B*44/B*40 148REYNEYENI IKZF3 B*44/B*49 149 SEKETVAYF INPP5F B*44 150 EEVTDRSQL IRF8B*44/B*40 151 EVDASIFKAW IRF8 B*44 152 AELLAKELY KIAA0226L B*44 153KEFEQVPGHL KIAA0226L B*44/B*40 154 AEPGPVITW LILRA4 B*44 155 NEFPVIVRLLRRK1 B*44 156 FEVESLFQKY MAP3K1 B*44 157 VEIAEAIQL MAP3K1 B*44/B*40 158GENEDNRIGL MCOLN2 B*44/B*40 159 GELLGRQSF MDM4 B*44 160 EEETILHFF NEK8B*44 161 EEGDTLLHLF NFKBID B*44 162 DEAQARAAF NLRP3 B*44 163 EEVVMGLLEYNLRP3 B*44 164 SEYSHLTRV NPR3 B*44/B*49 165 VELDLQRSV PIK3R6 B*44 166NEVLASKY PLCL2 B*44/B*18 167 KEIGAAVQAL PLEKHA2 B*44/B*40 168 QEIQSLLTNWPLEKHG1 B*44 169 EENGEVKEL PRDM2 B*44 170 SENEQRRMF PYHIN1 B*44 171SEDLAVHLY RALGPS2 B*44 172 VEDGLFHEF RBM15 B*44 173 KEYDFGTQL RNF220B*44/B*49 174 TDKSFPNAY RNGTT B*44/B*47 175 HEIDGKALFL SCML2 B*44 176AENAVSNLSF SLAMF6 B*44 177 QENMQIQSF SPG11 B*44 178 REYEHYWTEL STAP1B*44/B*50 179 AEIKQTEEKY VAV3 B*44 180 EEPAFNVSY VOPP1 B*44 181GEIKEPLEI VPS13C B*44/B*40/B*49 182 AQNLSIIQY WDFY4 B*44/B*15 183GESQDSTTAL WDFY4 B*44/B*40 184 RMPPFTQAF WDFY4 B*44/B*15 185 SEGDNVESWZCCHC7 B*44 186 NEQKIVRF ZNF699 B*44/B*18 187 SDAQRPSSF ZNF831 B*44/B*37

TABLE 2 Additional peptides according to the presentinvention with no prior known cancer  association. Seq Official ID GeneHLA No Sequence Symbol(s) allotype 188 YVDAGTPMY AGPAT5 A*01 189VTEEPQRLFY BMF A*01 190 HVDQDLTTY CDK6 A*01 191 ISEAGKDLLY CNTRL A*01192 RSDPGGGGLAY MEX3B A*01 193 LTDSEKGNSY RALGPS2 A*01 194 YTDKKSIIYSTAP1 A*01 195 YSDKEFAGSY TBC1D9 A*01 196 FTDIDGQVY WDR11 A*01 197SLADVHIEV BTAF1 A*02 198 KLLGYDVHV CASP2 A*02 199 AMPDSPAEV CBFA2T3 A*02200 VMLQINPKL CCDC88A A*02 201 ILAAVETRL FBXO28 A*02 202 MVALPMVLV ITGB7A*02 203 FLLPKVQSI KIAA0922 A*02 204 FLLPKVQSIQL KIAA0922 A*02 205FLINTNSEL PDE4A, A*02 PDE4B, PDE4C, PDE4D 206 SLMDLQERL STIM2 A*02 207KLSDNILKL SYNE2 A*02/B*13 208 KLNPQQAPLY AKAP13 A*03 209 KTLPAMLGTGKBTLA A*03/A*11 210 RMYSQLKTLQK DNMBP A*03 211 ATYNKQPMYR DNMT3A A*03 212LLWHWDTTQSLK FCER2 A*03 213 RVYNIYIRR GPR114 A*03/A*32 214 ATGAATPKKHIST1H1E A*03/A*11 215 KATGAATPK HIST1H1E A*03 216 RIKAPSRNTIQK MAP3K1A*03 217 TTVPHVFSK MAP3K1 A*03/A*11 218 RVLTGVFTK PARP15 A*03 219HSYSSPSTK RBM15 A*03 220 SISNLVFTY SOX4 A*03/A*29 221 LLNRHILAH ZNF669A*03 222 RYLDEINLL GNA15 A*24 223 RRMYPPPLI VOPP1 A*24/B*27 224VYEYVVERF ZCCHC11 A*24 225 LPARFYQAL AGPAT5 B*07 226 YLNRHLHTW BCL2B*07/A*32 227 APINKAGSFL BLK B*07 228 SPRITFPSL BLK B*07 229 SPLGSLARSSLCARD11 B*07 230 KPMKSVLVV CCR7 B*07 231 MPLSTIREV CDK6 B*07/B*51 232APRPAGSYL CIITA B*07 233 SPRVYWLGL CLEC17A B*07 234 SPKESENAL DEPDC5B*07 235 SPSLPSRTL DEPDC5 B*07 236 RPSNKAPLL EHMT1 B*07 237 SPWLHAPSLFAIM3 B*07 238 SPRSWIQVQI FCRL5 B*07 239 APSKTSLIM FOXP1 B*07 240SPSLPNITL HDAC4, B*07 HDAC9 241 APAPAEKTPV HIST1H1E B*07 242 SPFSFHHVLITGB7 B*07/B*35 243 LPKVQSIQL KIAA0922 B*07 244 MPSSDTTVTF MAP3K1B*07/B*35 245 SPLSHHSQL MAP3K1 B*07 246 YPGWHSTTI MYB B*07/B*51 247QPSPARAPAEL PMAIP1 B*07 248 LPYDSKHQI PTPN22 B*07/B*51 249 SPADHRGYASLSOX4 B*07 250 VPNLQTVSV SP4 B*07/B*51 251 QPRLFTMDL TRRAP B*07 252RPHIPISKL UBASH3B B*07 253 RPFADLLGTAF WDFY4 B*07 254 SPRNLQPQRAAL WDFY4B*07 255 YPGSDRIML WDFY4 B*07 256 SPYKKLKEAL TRAPPC10 B*08 257 KEFFFVKVFAIM2 B*44 258 EELFRDGVNW BCL2 B*44 259 EENTLVQNY BTAF1 B*44 260AEIGEGAYGKVF CDK6 B*44 261 NEIEHIPVW CNTRL B*44 262 QENQAETHAW CXCR5B*44 263 REAGFQVKAY FCRLA B*44 264 SEDHSGSYW FCRLA B*44 265 QEVDASIFKAWIRF8 B*44 266 VDASIFKAW IRF8 B*44 267 KEKFPINGW MTMR1 B*44 268 NEDKGTKAWMYSM1 B*44 269 KELEDLNKW PDE4B B*44 270 AESEDLAVHL RALGPS2 B*44/B*40 271AESEDLAVHLY RALGPS2 B*44 272 KEFELRSSW STIM2 B*44 273 AEIEIVKEEF SYNE2B*44 274 GEAVTDHPDRLW TCL1A B*44 275 TENPLTKLL TFEC B*44 276 EEEGNLLRSWWDFY4 B*44 277 EEGNLLRSW WDFY4 B*44

TABLE 3 Peptides useful for e.g. personalized  cancer therapies. SeqOfficial ID Gene HLA No Sequence Symbol(s) allotype 278 YLDRKLLTL SYKA*02 279 LYIDRPLPYL FAM21A, A*24 FAM21B, FAM21C

The present invention furthermore generally relates to the peptidesaccording to the present invention for use in the treatment ofproliferative diseases, such as, lymphoid neoplasms, for example,Non-Hodgkin lymphoma, post-transplant lymphoproliferative disorders(PTLD) for example as well as myeloid neoplasms, such as , primarymyelofibrosis, essential thrombocytopenia, polycythemia vera, as well asother neoplasms such as hepatocellular carcinoma, colorectal carcinoma,glioblastoma, gastric cancer, esophageal cancer, non-small cell lungcancer, small cell lung cancer, pancreatic cancer, renal cell carcinoma,prostate cancer, melanoma, breast cancer, gallbladder cancer andcholangiocarcinoma, urinary bladder cancer, uterine cancer, head andneck squamous cell carcinoma, mesothelioma.

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 279. More preferred are thepeptides—alone or in combination—selected from the group consisting ofSEQ ID NO: 1 to SEQ ID NO: 187 (see Table 1), and their uses in theimmunotherapy of chronic lymphocytic leukemia (CLL), acute myeloidleukemia (AML), chronic myeloid leukemia (CML) and other lymphoidneoplasms, for example, Non-Hodgkin lymphoma, post-transplantlymphoproliferative disorders (PTLD) as well as other myeloid neoplasms,such as primary myelofibrosis, essential thrombocytopenia, polycythemiavera, as well as other neoplasms such as hepatocellular carcinoma,colorectal carcinoma, glioblastoma, gastric cancer, esophageal cancer,non-small cell lung cancer, small cell lung cancer, pancreatic cancer,renal cell carcinoma, prostate cancer, melanoma, breast cancer,gallbladder cancer and cholangiocarcinoma, urinary bladder cancer,uterine cancer, head and neck squamous cell carcinoma, mesothelioma, andpreferably chronic lymphocytic leukemia, chronic myeloid leukemia andacute myeloid leukemia.

A particularly prefered combination of the peptides according to thepresent invention includes peptides presented by the seven most commonHLA-A and -B allotypes (se tables above), which allows for a medicamentproviding a (genetic) coverage of >92% of the European collective ofpatients.

Thus, another aspect of the present invention relates to the use of thepeptides according to the present invention for the—preferablycombined—treatment of a proliferative disease selected from the group ofchronic lymphocytic leukemia, chronic myeloid leukemia and acute myeloidleukemia, and other lymphoid neoplasms, for example, Non-Hodgkinlymphoma, post-transplant lymphoproliferative disorders (PTLD) as wellas other myeloid neoplasms, such as primary myelofibrosis, essentialthrombocytopenia, polycythemia vera, as well as other neoplasms such ashepatocellular carcinoma, colorectal carcinoma, glioblastoma, gastriccancer, esophageal cancer, non-small cell lung cancer, small cell lungcancer, pancreatic cancer, renal cell carcinoma, prostate cancer,melanoma, breast cancer, gallbladder cancer and cholangiocarcinoma,urinary bladder cancer, uterine cancer, head and neck squamous cellcarcinoma, mesothelioma.

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or—in an elongatedform, such as a length-variant—MHC class -II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 279.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofcancer.

The present invention further relates to antibodies that are specificagainst the peptides according to the present invention or complexes ofsaid peptides according to the present invention with MHC, and methodsof making these.

The present invention further relates to T-cell receptors (TCRs), inparticular soluble TCR (sTCRs) and cloned TCRs engineered intoautologous or allogeneic T cells, and methods of making these, as wellas NK cells or other cells bearing said TCR or cross-reacting with saidTCRs.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to the invention athand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing or expressing said peptidecontaining SEQ ID No. 1 to SEQ ID No.: 279, preferably containing SEQ IDNo. 1 to SEQ ID No. 187, or a variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed, the nucleic acid according to the present invention, theexpression vector according to the present invention, the cell accordingto the present invention, the activated T lymphocyte, the T cellreceptor or the antibody or other peptide- and/or peptide-MHC-bindingmolecules according to the present invention as a medicament or in themanufacture of a medicament. Preferably, said medicament is activeagainst cancer.

Preferably, said medicament is a cellular therapy, a vaccine or aprotein based on a soluble TCR or antibody.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are chronic lymphocytic leukemia,chronic myeloid leukemia and acute myeloid leukemia, and other lymphoidneoplasms, for example, Non-Hodgkin lymphoma, post-transplantlymphoproliferative disorders (PTLD) as well as other myeloid neoplasms,such as primary myelofibrosis, essential thrombocytopenia, polycythemiavera, as well as other neoplasms such as hepatocellular carcinoma,colorectal carcinoma, glioblastoma, gastric cancer, esophageal cancer,non-small cell lung cancer, small cell lung cancer, pancreatic cancer,renal cell carcinoma, prostate cancer, melanoma, breast cancer,gallbladder cancer and cholangiocarcinoma, urinary bladder cancer,uterine cancer, head and neck squamous cell carcinoma, mesothelioma, andpreferably chronic lymphocytic leukemia, chronic myeloid leukemia andacute myeloid leukemia cells.

The present invention further relates to biomarkers based on thepeptides according to the present invention, herein called “targets”that can be used in the diagnosis of cancer, preferably chroniclymphocytic leukemia, chronic myeloid leukemia and acute myeloidleukemia. The marker can be over-presentation of the peptide(s)themselves, or over-expression of the corresponding gene(s). The markersmay also be used to predict the probability of success of a treatment,preferably an immunotherapy, and most preferred an immunotherapytargeting the same target that is identified by the biomarker. Forexample, an antibody or soluble TCR can be used to stain sections of thetumor to detect the presence of a peptide of interest in complex withMHC.

Optionally the antibody carries a further effector function such as animmune stimulating domain or toxin.

The present invention also relates to the use of these novel targets inthe context of cancer treatment.

DETAILED DESCRIPTION OF THE INVENTION

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, or 12 or longer, and incase of MHC class II peptides (elongated variants of the peptides of theinvention) they can be as long as 13, 14, 15, 16, 17, 18, 19 or 20 ormore amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the invention, as long asthe correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans, there are three different genetic loci that encode MHC classI molecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

TABLE 4 Expression frequencies F of HLA-A*02, HLA-A*01, HLA-A*03, HLA-A*24, HLA-B*07, HLA-B*08 and HLA-B*44 serotypes. Haplotype frequenciesGf are derived from a study which used HLA-typing data from a registryof more than 6.5 million volunteer donors in the U.S. (Gragert et al.,2013). The haplotype frequency is the frequency of a distinct allele onan individual chromosome. Due to the diploid set of chromosomes withinmammalian cells, the frequency of genotypic occurrence of this allele ishigher and can be calculated employing the Hardy-Weinberg principle (F =1 − (1 − Gf)²). Calculated phenotype from Allele Population allelefrequency (F) A*02 African (N = 28557) 32.3% European Caucasian 49.3% (N= 1242890) Japanese (N =24582) 42.7% Hispanic, S + Cent Amer. 46.1% (N =146714) Southeast Asian (N = 27978) 30.4% A*01 African (N = 28557) 10.2%European Caucasian 30.2% (N = 1242890) Japanese (N = 24582) 1.8%Hispanic, S + Cent Amer. 14.0% (N = 146714) Southeast Asian (N = 27978)21.0% A*03 African (N = 28557) 14.8% European Caucasian 26.4% (N =1242890) Japanese (N = 24582) 1.8% Hispanic, S + Cent Amer. 14.4% (N =146714) Southeast Asian (N = 27978) 10.6% A*24 African (N = 28557) 2.0%European Caucasian 8.6% (N = 1242890) Japanese (N = 24582) 35.5%Hispanic, S + Cent Amer. 13.6% (N = 146714) Southeast Asian (N = 27978)16.9% B*07 African (N = 28557) 14.7% European Caucasian 25.0% (N =1242890) Japanese (N = 24582) 11.4% Hispanic, S + Cent Amer. 12.2% (N =146714) Southeast Asian (N = 27978) 10.4% B*08 African (N = 28557) 6.0%European Caucasian 21.6% (N = 1242890) Japanese (N = 24582) 1.0%Hispanic, S + Cent Amer. 7.6% (N = 146714) Southeast Asian (N = 27978)6.2% B*44 African (N = 28557) 10.6% European Caucasian 26.9% (N =1242890) Japanese (N = 24582) 13.0% Hispanic, S + Cent Amer. 18.2% (N =146714) Southeast Asian (N = 27978) 13.1%

The peptides of the invention, preferably when included into a vaccineof the invention as described herein bind to A*02, A*01, A*03, A*24,B*07, B*08 or B*44. A vaccine may also include pan-binding MHC class IIpeptides. Therefore, the vaccine of the invention can be used to treatcancer in patients that are A*02-, A*01-, A*03-, A*24-, B*07-, B*08- orB*44-positive, whereas no selection for MHC class II allotypes isnecessary due to the pan-binding nature of these peptides.

If A*02 peptides of the invention are combined with peptides binding toanother allele, for example A*24, a higher percentage of any patientpopulation can be treated compared with addressing either MHC class Iallele alone. While in most populations less than 50% of patients couldbe addressed by either allele alone, a vaccine comprising HLA-A*24 andHLA-A*02 epitopes can treat at least 60% of patients in any relevantpopulation. Specifically, the following percentages of patients will bepositive for at least one of these alleles in various regions: USA 61%,Western Europe 62%, China 75%, South Korea 77%, Japan 86% (calculatedfrom www.allelefrequencies.net).

TABLE 5 HLA alleles coverage in European Caucasian population(calculated from (Gragert et al., 2013)). coverage (at least combinedone A- combined combined with B*07 allele) with B*07 with B*44 and B*44A*02/A*01 70% 78% 78% 84% A*02/A*03 68% 76% 76% 83% A*02/A*24 61% 71%71% 80% A*01/A*03 52% 64% 65% 75% A*01/A*24 44% 58% 59% 71% A*03/A*2440% 55% 56% 69% A*02/A*01/A*03 84% 88% 88% 91% A*02/A*01/A*24 79% 84%84% 89% A*02/A*03/A*24 77% 82% 83% 88% A*01/A*03/A*24 63% 72% 73% 81%A*02/A*01/A*03/A*24 90% 92% 93% 95%

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The percent identity isthen determined according to the following formula:

percent identity=100[1−(C/R)]

wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein

(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and

(ii) each gap in the Reference Sequence and

(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and

(iiii) the alignment has to start at position 1 of the alignedsequences;

and R is the number of bases or amino acids in the Reference Sequenceover the length of the alignment with the Compared Sequence with any gapcreated in the Reference Sequence also being counted as a base or aminoacid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 279 or a variant thereof which is 88%homologous to SEQ ID NO: 1 to SEQ ID NO: 279, or a variant thereof thatwill induce T cells cross-reacting with said peptide. The peptides ofthe invention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or elongated versions of saidpeptides to class II.

In the present invention, the term “homologous” refers to the degree ofidentity (see percent identity above) between sequences of two aminoacid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 279. Forexample, a peptide may be modified so that it at least maintains, if notimproves, the ability to interact with and bind to the binding groove ofa suitable MHC molecule, such as HLA-A*02 or -DR, and in that way, it atleast maintains, if not improves, the ability to bind to the TCR ofactivated T cells.

These T cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide as defined in the aspects of the invention. Ascan be derived from the scientific literature and databases (Rammenseeet al., 1999; Godkin et al., 1997), certain positions of HLA bindingpeptides are typically anchor residues forming a core sequence fittingto the binding motif of the HLA receptor, which is defined by polar,electrophysical, hydrophobic and spatial properties of the polypeptidechains constituting the binding groove. Thus, one skilled in the artwould be able to modify the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO 279, by maintaining the known anchor residues, and wouldbe able to determine whether such variants maintain the ability to bindMHC class I or II molecules. The variants of the present inventionretain the ability to bind to the TCR of activated T cells, which cansubsequently cross-react with and kill cells that express a polypeptidecontaining the natural amino acid sequence of the cognate peptide asdefined in the aspects of the invention.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective, since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,non-standard amino acids (i.e., other than the common naturallyoccurring proteinogenic amino acids) may also be used for substitutionpurposes to produce immunogens and immunogenic polypeptides according tothe present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without that the ability to bind to amolecule of the human major histocompatibility complex (MHC) class-I or-II is substantially changed or is negatively affected, when compared tothe non-modified peptide. In another embodiment, in a peptide consistingessentially of the amino acid sequence as indicated herein, one or twoamino acids can be exchanged with their conservative exchange partners(see herein below) without that the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or -II issubstantially changed, or is negatively affected, when compared to thenon-modified peptide.

The amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith other amino acid whose incorporation does not substantially affectT-cell reactivity and does not eliminate binding to the relevant MHC.Thus, apart from the proviso given, the peptide of the invention may beany peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 6 Variants and motif of the peptides  according to SEQ ID NO: 1, 193, 17,   27, 33, 210, 64, 73, 99, 238, 116,   118, 134 and 148. Position 1 2 3 45 6 7 8 9 10 11 SEQ ID L T E G H S G N Y Y No 1 Variant S D S D A S S AD D A A SEQ ID L T D S E K G N S Y No 193 Variant S S A S E S E A A E EA SEQ ID K A Y N R V I F V No 17 Variant L L I L L L A M M I M L M A I LA V V I V L V A T T I T L T A Q Q I Q L Q A SEQ ID R L I A K E M N INo 27 Variant V L A M V M M L M A A V A A L A A V V V V L V A T V T T LT A Q V Q Q L Q A SEQ ID S V F E G D S I V L K No 33 Variant L L Y L R LF I I Y I R I F M M Y M R M F Y R F T T Y T R T F SEQ ID R M Y S Q L K TL Q K No 210 Variant L L Y L R L F I I Y I R I F Y R F V V Y V R V F T TY T R T F SEQ ID S F Q S K A T V F No 64 Variant Y I Y L Y I L SEQ ID TY P Q L E G F K F No 73 Variant I L F I F L F SEQ ID S P R A I N N L V LNo 99 Variant F V M A I SEQ ID S P R S W I Q V Q I No 238 Variant L F VM A SEQ ID T L K I R A E V L No 116 Variant K K V K I K M K F V I M F HH V H I H M H F R K R K V R K I R K M R K F R R V R I R M R F R H R H VR H I R H M R H F L K L K V L K I L K M L K F L L V L I L M L F L H L HV L H I L H M L H F SEQ ID F E K E K K E S L No 118 Variant V I M F R RV R I R M R F H H V H I H M H F R R V R I R M R F R R R R V R R I R R MR R F R H R H V R H I R H M R H F L L V L I L M L F L R L R V L R I L RM L R F L H L H V L H I L H M L H F SEQ ID S E Y A D T H Y F No 134Variant W Y L D D W D Y D L SEQ ID R E Y N E Y E N I No 148 Variant F WY L D F D W D Y D L

Longer (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually between 8 and 11 amino acidslong, are generated by peptide processing from longer peptides orproteins that include the actual epitope. It is preferred that theresidues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4. Combinations of the elongationsaccording to the invention can be found in Table 7.

TABLE 7 Combinations of the elongations of peptides of the inventionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than 4 amino acids, preferably to a total length of up to30 amino acids. This may lead to MHC class II binding peptides. Bindingto MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof between 8 and 100, preferably between 8 and 30, and most preferredbetween 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in caseof the elongated class II binding peptides the length can also be 15,16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art.

Preferably, when the T cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 pM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 pM, andmost preferably no more than about 10 pM. It is also preferred that thesubstituted peptide be recognized by T cells from more than oneindividual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 279.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO 279 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “Ii”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond, amino acid residues are not joined by peptide(-CO-NH-) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) (Meziere et al., 1997),incorporated herein by reference. This approach involves makingpseudopeptides containing changes involving the backbone, and not theorientation of side chains. Meziere et al. (Meziere et al., 1997) showthat for MHC binding and T helper cell responses, these pseudopeptidesare useful. Retro-inverse peptides, which contain NH—CO bonds instead ofCO—NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH=CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which isincorporated herein by reference. Chemical modification of amino acidsincludes but is not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995)for more extensive methodology relating to chemical modification ofproteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich(http://www.sigma-aldrich.com) provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide can be used to form intra-molecular crosslinks between alysine residue and a glutamic acid residue. For example,diethylpyrocarbonate is a reagent for the modification of histidylresidues in proteins. Histidine can also be modified using4-hydroxy-2-nonenal. The reaction of lysine residues and other α-aminogroups is, for example, useful in binding of peptides to surfaces or thecross-linking of proteins/peptides. Lysine is the site of attachment ofpoly(ethylene)glycol and the major site of modification in theglycosylation of proteins. Methionine residues in proteins can bemodified with e.g. iodoacetamide, Bromo ethylamine, and chloramine T.

Tetranitromethane and N-acetyl imidazole can be used for themodification of tyrosyl residues. Cross-linking via the formation ofdityrosine can be accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention.

Another embodiment of the present invention relates to a non-naturallyoccurring peptide wherein said peptide consists or consists essentiallyof an amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 279and has been synthetically produced (e.g. synthesized) as apharmaceutically acceptable salt. Methods to synthetically producepeptides are well known in the art. The salts of the peptides accordingto the present invention differ substantially from the peptides in theirstate(s) in vivo, as the peptides as generated in vivo are no salts. Thenon-natural salt form of the peptide mediates the solubility of thepeptide, in particular in the context of pharmaceutical compositionscomprising the peptides, e.g. the peptide vaccines as disclosed herein.A sufficient and at least substantial solubility of the peptide(s) isrequired in order to efficiently provide the peptides to the subject tobe treated. Preferably, the salts are pharmaceutically acceptable saltsof the peptides. These salts according to the invention include alkalineand earth alkaline salts such as salts of the Hofmeister seriescomprising as anions PO₄ ³⁻, SO₄ ²⁻, CH₃COO⁻, Cl⁻, Br⁻, NO₃ ⁻, ClO₄ ⁻,SCN⁻and as cations NH₄ ⁺, Rb⁺, K⁺, Na⁺, Cs⁺, Li⁺, Zn²⁺, Mg²⁺,Ca^(2hu +), Mn²⁺, Cu²⁺ and Ba²⁺. Particularly salts are selected from(NH₄)₃PO₄, (NH₄)₂HPO₄, (NH₄)H₂PO₄, (NH₄)₂SO₄, NH₄CH₃COO, NH₄Cl, NH₄Br,NH₄NO₃, NH₄ClO₄, NH₄I, NH₄SCN, Rb₃PO₄, Rb₂HPO₄, RbH₂PO₄, Rb₂SO₄,Rb₄CH₃COO, Rb₄Cl, Rb₄Br, Rb₄NO₃, Rb₄ClO₄, Rb₄I, Rb₄SCN, K₃PO₄, K₂HPO₄,KH₂PO₄, K₂SO₄, KCH₃COO, KCl, KBr, KNO₃, KCIO₄, KI, KSCN, Na₃PO₄,Na₂HPO₄, NaH₂PO₄, Na₂SO₄, NaCH₃COO, NaCl, NaBr, NaNO₃, NaClO₄, NaI,NaSCN, ZnCl₂ Cs₃PO₄, Cs₂HPO₄, CsH₂PO₄, Cs₂SO₄, CsCH₃COO, CsCl, CsBr,CsNO₃, CsClO₄, CSI, CsSCN, Li₃PO₄, Li₂HPO₄, LiH₂PO₄, Li₂SO₄, LiCH₃COO,LiCl, LiBr, LiNO₃, LiClO₄, LiI, LiSCN, Cu₂SO₄, Mg₃(PO₄)₂, Mg₂HPO₄,Mg(H₂PO₄)₂, Mg₂SO₄, Mg(CH₃COO)₂, MgCl₂, MgBr₂, Mg(NO₃)₂, Mg(ClO₄)₂,MgI₂, Mg(SCN)₂, MnCl₂, Ca₃(PO₄), Ca₂HPO₄, Ca(H₂PO₄)₂, CaSO₄,Ca(CH₃COO)₂, CaCl₂, CaBr₂, Ca(NO₃)₂, Ca(ClO₄)₂, CaI₂, Ca(SCN)₂,Ba₃(PO₄)₂, Ba₂HPO₄, Ba(H₂PO₄)₂, BaSO₄, Ba(CH₃COO)₂, BaCl₂, BaBr₂,Ba(NO₃)₂, Ba(CIO₄)₂, BaI₂, and Ba(SCN)₂. Particularly preferred are NHacetate, MgCl₂, KH₂PO₄, Na₂SO₄, KCl, NaCl, and CaCl₂, such as, forexample, the chloride or acetate (trifluoroacetate) salts.

Generally, peptides and variants (at least those containing peptidelinkages between amino acid residues) may be synthesized by theFmoc-polyamide mode of solid-phase peptide synthesis as disclosed byLukas et al. (Lukas et al., 1981) and by references as cited therein.Temporary N-amino group protection is afforded by the9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of thishighly base-labile protecting group is done using 20% piperidine in N,N-dimethylformamide. Side-chain functionalities may be protected astheir butyl ethers (in the case of serine threonine and tyrosine), butylesters (in the case of glutamic acid and aspartic acid),butyloxycarbonyl derivative (in the case of lysine and histidine),trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethyl acrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N, N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated coupling procedure. All coupling anddeprotection reactions are monitored using ninhydrin, trinitrobenzenesulphonic acid or isotin test procedures. Upon completion of synthesis,peptides are cleaved from the resin support with concomitant removal ofside-chain protecting groups by treatment with 95% trifluoracetic acidcontaining a 50% scavenger mix. Scavengers commonly used includeethanedithiol, phenol, anisole and water, the exact choice depending onthe constituent amino acids of the peptide being synthesized. Also acombination of solid phase and solution phase methodologies for thesynthesis of peptides is possible (see, for example, (Bruckdorfer etal., 2004), and the references as cited therein).

Trifluoracetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated and sequences were identified by onlinenano-electrospray-ionization (nanoESI) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of naturaltumor-associated peptides (TUMAPs) recorded from chronic lymphocyticleukemia (N=35 samples), chronic myeloid leukemia (N=16 samples) andacute myeloid leukemia (N=32 samples) samples with the fragmentationpatterns of corresponding synthetic reference peptides of identicalsequences. Since the peptides were directly identified as ligands of HLAmolecules of primary tumors, these results provide direct evidence forthe natural processing and presentation of the identified peptides onprimary cancer tissue obtained from 83 chronic lymphocytic leukemia,chronic myeloid leukemia and acute myeloid leukemia patients (cf.Example 1).

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US2013-0096016, which is hereby incorporated by reference in its entirety)allows the identification and selection of relevant over-presentedpeptide vaccine candidates based on direct relative quantitation ofHLA-restricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

HLA-peptide complexes from chronic lymphocytic leukemia, chronic myeloidleukemia and acute myeloid leukemia tissue samples were purified andHLA-associated peptides were isolated and analyzed by LC-MS (see example1). All TUMAPs contained in the present application were identified withthis approach on primary chronic lymphocytic leukemia, chronic myeloidleukemia and acute myeloid leukemia samples confirming theirpresentation on primary chronic lymphocytic leukemia, chronic myeloidleukemia and acute myeloid leukemia.

Besides presentation of the peptide, mRNA expression of the underlyinggene was tested. mRNA data were obtained via RNASeq analyses of normaltissues and cancer tissues (cf. Example 2, FIG. 1). Peptides which arederived from proteins whose coding mRNA is highly expressed in cancertissue, but very low or absent in vital normal tissues, were preferablyincluded in the present invention.

The present invention provides peptides that are useful in treatingcancers/tumors, preferably chronic lymphocytic leukemia, chronic myeloidleukemia and acute myeloid leukemia that over- or exclusively presentthe peptides of the invention. These peptides were shown by massspectrometry to be naturally presented by HLA molecules on primary humanchronic lymphocytic leukemia, chronic myeloid leukemia and acute myeloidleukemia samples.

Many of the source gene/proteins (also designated “full-length proteins”or “underlying proteins”) from which the peptides are derived were shownto be highly over-expressed in cancer compared with normaltissues—“normal tissues” in relation to this invention shall mean eitherhealthy peripheral blood mononuclear cells (PBMC) cells or other normaltissue cells, demonstrating a high degree of tumor association of thesource genes (see Example 2). Moreover, the peptides themselves arepresented on tumor tissue—“tumor tissue” in relation to this inventionshall mean a sample from a patient suffering from chronic lymphocyticleukemia, chronic myeloid leukemia and acute myeloid leukemia.

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes. T cells can destroy the cells presenting the recognizedHLA/peptide complex, e.g. chronic lymphocytic leukemia, chronic myeloidleukemia and acute myeloid leukemia cells presenting the derivedpeptides.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, such as soluble TCRs,according to the present invention (see Example 3, Example 4).Furthermore, the peptides when complexed with the respective MHC can beused for the production of antibodies and/or TCRs, in particular sTCRs,according to the present invention, as well. Respective methods are wellknown to the person of skill, and can be found in the respectiveliterature as well (see also below). Thus, the peptides of the presentinvention are useful for generating an immune response in a patient bywhich tumor cells can be destroyed. An immune response in a patient canbe induced by direct administration of the described peptides orsuitable precursor substances (e.g. elongated peptides, proteins, ornucleic acids encoding these peptides) to the patient, ideally incombination with an agent enhancing the immunogenicity (i.e. anadjuvant). The immune response originating from such a therapeuticvaccination can be expected to be highly specific against tumor cellsbecause the target peptides of the present invention are not presentedon normal tissues in comparable copy numbers, preventing the risk ofundesired autoimmune reactions against normal cells in the patient.

The present description further relates to T-cell receptors (TCRs)comprising an alpha chain and a beta chain (“alpha/beta TCRs”). Alsoprovided are peptides according to the invention capable of binding toTCRs and antibodies when presented by an MHC molecule.

The present description also relates to fragments of the TCRs accordingto the invention that are capable of binding to a peptide antigenaccording to the present invention when presented by an HLA molecule.The term particularly relates to soluble TCR fragments, for example TCRsmissing the transmembrane parts and/or constant regions, single chainTCRs, and fusions thereof to, for example, with Ig.

The present description also relates to nucleic acids, vectors and hostcells for expressing TCRs and peptides of the present description; andmethods of using the same.

The term “T-cell receptor” (abbreviated TCR) refers to a heterodimericmolecule comprising an alpha polypeptide chain (alpha chain) and a betapolypeptide chain (beta chain), wherein the heterodimeric receptor iscapable of binding to a peptide antigen presented by an HLA molecule.The term also includes so-called gamma/delta TCRs.

In one embodiment, the description provides a method of producing a TCRas described herein, the method comprising culturing a host cell capableof expressing the TCR under conditions suitable to promote expression ofthe TCR.

The description in another aspect relates to methods according to thedescription, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell or the antigen is loadedonto class I or II MHC tetramers by tetramerizing the antigen/class I orII MHC complex monomers.

The alpha and beta chains of alpha/beta TCR's, and the gamma and deltachains of gamma/delta TCRs, are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region (V), and joining region(J). The variable domain may also include a leader region (L). Beta anddelta chains may also include a diversity region (D). The alpha and betaconstant domains may also include C-terminal transmembrane (TM) domainsthat anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term “TCR gamma variable domain”as used herein refers to the concatenation of the TCR gamma V (TRGV)region without leader region (L), and the TCR gamma J (TRGJ) region, andthe term TCR gamma constant domain refers to the extracellular TRGCregion, or to a C-terminal truncated TRGC sequence. Likewise, the term“TCR delta variable domain” refers to the concatenation of the TCR deltaV (TRDV) region without leader region (L) and the TCR delta D/J(TRDD/TRDJ) region, and the term “TCR delta constant domain” refers tothe extracellular TRDC region, or to a C-terminal truncated TRDCsequence.

TCRs of the present description preferably bind to a peptide-HLAmolecule complex with a binding affinity (KD) of about 100 μM or less,about 50 μM or less, about 25 μM or less, or about 10 μM or less. Morepreferred are high affinity TCRs having binding affinities of about 1 μMor less, about 100 nM or less, about 50 nM or less, about 25 nM or less.Non-limiting examples of preferred binding affinity ranges for TCRs ofthe present invention include about 1 nM to about 10 nM; about 10 nM toabout 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40 nM;about 40 nM to about 50 nM; about 50 nM to about 60 nM; about 60 nM toabout 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM; andabout 90 nM to about 100 nM.

As used herein in connect with TCRs of the present description,“specific binding” and grammatical variants thereof are used to mean aTCR having a binding affinity (KD) for a peptide-HLA molecule complex of100 μM or less.

Alpha/beta heterodimeric TCRs of the present description may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned above,alpha/beta hetero-dimeric TCRs of the present description may have aTRAC constant domain sequence and a TRBC1 or TRBC2 constant domainsequence, and the TRAC constant domain sequence and the TRBC1 or TRBC2constant domain sequence of the TCR may be linked by the nativedisulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 ofTRBC1 or TRBC2.

TCRs of the present description may comprise a detectable label selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.TCRs of the present description may be conjugated to a therapeuticallyactive agent, such as a radionuclide, a chemotherapeutic agent, or atoxin.

In an embodiment, a TCR of the present description having at least onemutation in the alpha chain and/or having at least one mutation in thebeta chain has modified glycosylation compared to the unmutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCRalpha chain and/or TCR beta chain has a binding affinity for, and/or abinding half-life for, a peptide-HLA molecule complex, which is at leastdouble that of a TCR comprising the unmutated TCR alpha chain and/orunmutated TCR beta chain. Affinity-enhancement of tumor-specific TCRs,and its exploitation, relies on the existence of a window for optimalTCR affinities. The existence of such a window is based on observationsthat TCRs specific for HLA-A2-restricted pathogens have KD values thatare generally about 10-fold lower when compared to TCRs specific forHLA-A2-restricted tumor-associated self-antigens. It is now known,although tumor antigens have the potential to be immunogenic, becausetumors arise from the individual's own cells only mutated proteins orproteins with altered translational processing will be seen as foreignby the immune system. Antigens that are upregulated or overexpressed (socalled self-antigens) will not necessarily induce a functional immuneresponse against the tumor: T-cells expressing TCRs that are highlyreactive to these antigens will have been negatively selected within thethymus in a process known as central tolerance, meaning that onlyT-cells with low-affinity TCRs for self-antigens remain. Therefore,affinity of TCRs or variants of the present description to peptides canbe enhanced by methods well known in the art.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*02-negative healthy donors withA2/peptide monomers, incubating the PBMCs with tetramer-phycoerythrin(PE) and isolating the high avidity T-cells by fluorescence activatedcell sorting (FACS)—Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRαβ geneloci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with a peptide, incubating PBMCs obtained from the transgenic micewith tetramer-phycoerythrin (PE), and isolating the high avidity T-cellsby fluorescence activated cell sorting (FACS)—Calibur analysis.

In one aspect, to obtain T-cells expressing TCRs of the presentdescription, nucleic acids encoding TCR-alpha and/or TCR-beta chains ofthe present description are cloned into expression vectors, such asgamma retrovirus or lentivirus. The recombinant viruses are generatedand then tested for functionality, such as antigen specificity andfunctional avidity. An aliquot of the final product is then used totransduce the target T-cell population (generally purified from patientPBMCs), which is expanded before infusion into the patient.

In another aspect, to obtain T-cells expressing TCRs of the presentdescription, TCR RNAs are synthesized by techniques known in the art,e.g., in vitro transcription systems. The in vitro-synthesized TCR RNAsare then introduced into primary CD8+ T-cells obtained from healthydonors by electroporation to re-express tumor specific TCR-alpha and/orTCR-beta chains.

To increase the expression, nucleic acids encoding TCRs of the presentdescription may be operably linked to strong promoters, such asretroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murinestem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin,ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter,elongation factor (EF)-1a and the spleen focus-forming virus (SFFV)promoter. In a preferred embodiment, the promoter is heterologous to thenucleic acid being expressed.

In addition to strong promoters, TCR expression cassettes of the presentdescription may contain additional elements that can enhance transgeneexpression, including a central polypurine tract (cPPT), which promotesthe nuclear translocation of lentiviral constructs (Follenzi et al.,2000), and the woodchuck hepatitis virus posttranscriptional regulatoryelement (wPRE), which increases the level of transgene expression byincreasing RNA stability (Zufferey et al., 1999).

The alpha and beta chains of a TCR of the present invention may beencoded by nucleic acids located in separate vectors, or may be encodedby polynucleotides located in the same vector.

Achieving high-level TCR surface expression requires that both theTCR-alpha and TCR-beta chains of the introduced TCR be transcribed athigh levels. To do so, the TCR-alpha and TCR-beta chains of the presentdescription may be cloned into bi-cistronic constructs in a singlevector, which has been shown to be capable of over-coming this obstacle.The use of a viral intraribosomal entry site (IRES) between theTCR-alpha and TCR-beta chains results in the coordinated expression ofboth chains, because the TCR-alpha and TCR-beta chains are generatedfrom a single transcript that is broken into two proteins duringtranslation, ensuring that an equal molar ratio of TCR-alpha andTCR-beta chains are produced (Schmitt et al., 2009).

Nucleic acids encoding TCRs of the present description may be codonoptimized to increase expression from a host cell. Redundancy in thegenetic code allows some amino acids to be encoded by more than onecodon, but certain codons are less “optimal” than others because of therelative availability of matching tRNAs as well as other factors(Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta genesequences such that each amino acid is encoded by the optimal codon formammalian gene expression, as well as eliminating mRNA instabilitymotifs or cryptic splice sites, has been shown to significantly enhanceTCR-alpha and TCR-beta gene expression (Scholten et al., 2006).

Furthermore, mispairing between the introduced and endogenous TCR chainsmay result in the acquisition of specificities that pose a significantrisk for autoimmunity. For example, the formation of mixed TCR dimersmay reduce the number of CD3 molecules available to form properly pairedTCR complexes, and therefore can significantly decrease the functionalavidity of the cells expressing the introduced TCR (Kuball et al.,2007).

To reduce mispairing, the C-terminus domain of the introduced TCR chainsof the present description may be modified in order to promoteinterchain affinity, while de-creasing the ability of the introducedchains to pair with the endogenous TCR. These strategies may includereplacing the human TCR-alpha and TCR-beta C-terminus domains with theirmurine counterparts (murinized C-terminus domain); generating a secondinterchain disulfide bond in the C-terminus domain by introducing asecond cysteine residue into both the TCR-alpha and TCR-beta chains ofthe introduced TCR (cysteine modification); swapping interactingresidues in the TCR-alpha and TCR-beta chain C-terminus domains(“knob-in-hole”); and fusing the variable domains of the TCR-alpha andTCR-beta chains directly to CD3 (CD3 fusion) (Schmitt et al., 2009).

In an embodiment, a host cell is engineered to express a TCR of thepresent description. In preferred embodiments, the host cell is a humanT-cell or T-cell progenitor. In some embodiments, the T-cell or T-cellprogenitor is obtained from a cancer patient. In other embodiments, theT-cell or T-cell progenitor is obtained from a healthy donor. Host cellsof the present description can be allogeneic or autologous with respectto a patient to be treated. In one embodiment, the host is a gamma/deltaT-cell transformed to express an alpha/beta TCR.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH2 group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is animmunotherapeutic such as a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically i.d., i.m.,s.c., i.p. and i.v., or applied ex vivo to cells derived from thepatient or a human cell line which are subsequently administered to thepatient, or used in vitro to select a subpopulation of immune cellsderived from the patient, which are then re-administered to the patient.If the nucleic acid is administered to cells in vitro, it may be usefulfor the cells to be transfected so as to co-express immune-stimulatingcytokines, such as interleukin-2. The peptide may be substantially pure,or combined with an immune-stimulating adjuvant (see below) or used incombination with immune-stimulatory cytokines, or be administered with asuitable delivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T cells. However, stimulation ofCD8 T cells is more efficient in the presence of help provided by CD4T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8 Tcells the fusion partner or sections of a hybrid molecule suitablyprovide epitopes which stimulate CD4-positive T cells. CD4- andCD8-stimulating epitopes are well known in the art and include thoseidentified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 279, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. Thepeptide(s) may be derived from one or more specific TAAs and may bind toMHC class I molecules.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA.

The vector and DNA segment are then joined by hydrogen bonding betweenthe complementary homopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by SaikiRK, et al. (Saiki et al., 1988). This method may be used for introducingthe DNA into a suitable vector, for example by engineering in suitablerestriction sites, or it may be used to modify the DNA in other usefulways as is known in the art. If viral vectors are used, pox- oradenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Ylps) and incorporate the yeast selectable markers HIS3, TRP1, LEU2and URA3. Plasm ids pRS413-416 are Yeast Centromere plasmids (Ycps). CMVpromoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3xFLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL, or may be linked without anyadditional peptide(s) between them. These constructs can also be usedfor cancer therapy, and may induce immune responses both involving MHC Iand MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343).

Preferred eukaryotic host cells include yeast, insect and mammaliancells, preferably vertebrate cells such as those from a mouse, rat,monkey or human fibroblastic and colon cell lines. Yeast host cellsinclude YPH499, YPH500 and YPH501, which are generally available fromStratagene Cloning Systems, La Jolla, CA 92037, USA. Preferred mammalianhost cells include Chinese hamster ovary (CHO) cells available from theATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from theATCC as CRL 1658, monkey kidney-derived COS-1 cells available from theATCC as CRL 1650 and 293 cells which are human embryonic kidney cells.Preferred insect cells are Sf9 cells which can be transfected withbaculovirus expression vectors. An overview regarding the choice ofsuitable host cells for expression can be found in, for example, thetextbook of Paulina Balbás and Argelia Lorence “Methods in MolecularBiology Recombinant Gene Expression, Reviews and Protocols,” Part One,Second Edition, ISBN 978-1-58829-262-9, and other literature known tothe person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (Cohen et al.,1972) and (Green and Sambrook, 2012) . Transformation of yeast cells isdescribed in Sherman et al. (Sherman et al., 1986) . The method of Beggs(Beggs, 1978) is also useful. With regard to vertebrate cells, reagentsuseful in transfecting such cells, for example calcium phosphate andDEAE-dextran or liposome formulations, are available from StratageneCloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877,USA. Electroporation is also useful for transforming and/or transfectingcells and is well known in the art for transforming yeast cell,bacterial cells, insect cells and vertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment, the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment, the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantiallypure, or contained in a suitable vector or delivery system. The nucleicacid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g. Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CD8-positive T cellsand helper-T (TH) cells to an antigen, and would thus be considereduseful in the medicament of the present invention. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligandsderived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod(ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, JuvImmune®, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system,poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also, cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC),poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, Bevacizumab®, Celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodiestargeting key structures of the immune system (e.g. anti-CD40,anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may acttherapeutically and/or as an adjuvant. The amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan without undueexperimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivates, poly-(I:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Thepeptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomatous or cancerous diseases. Exemplary formulations can be foundin, for example, EP2112253.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore, differentcancer associated signaling pathways are attacked. This is an advantageover vaccines that address only one or few targets, which may cause thetumor to easily adapt to the attack (tumor escape). Furthermore, not allindividual tumors express the same pattern of antigens. Therefore, acombination of several tumor-associated peptides ensures that everysingle tumor bears at least some of the targets. The composition isdesigned in such a way that each tumor is expected to express several ofthe antigens and cover several independent pathways necessary for tumorgrowth and maintenance. Thus, the vaccine can easily be used“off-the-shelf” for a larger patient population. This means that apre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g. antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g. a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g. the complex of a peptide with MHC, accordingto the application at hand). In another embodiment, a scaffold is ableto activate signaling through its target antigen, for example a T cellreceptor complex antigen. Scaffolds include but are not limited toantibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complexof interest better than other naturally occurring peptide-MHC-complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting another peptide-MHCcomplex(es). Binding to other peptide-MHC complexes is irrelevant if thepeptide of the cross-reactive peptide-MHC is not naturally occurring,i.e. not derived from the human HLA-peptidome. Tests to assess targetcell killing are well known in the art. They should be performed usingtarget cells (primary cells or cell lines) with unaltered peptide-MHCpresentation, or cells loaded with peptides such that naturallyoccurring peptide-MHC levels are reached.

Each scaffold can comprise a labelling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labelledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example, afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualization of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, and anti-CD28.

For further information on polypeptide scaffolds see for example thebackground section of WO 2014/071978A1 and the references cited therein.

The present invention further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable alternatives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, itcould be shown that some of the aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides comprising, preferably consistingof, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 279,according to the invention at hand with the MHC molecule, using thecell-SELEX (Systematic Evolution of Ligands by Exponential enrichment)technique.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen (preferably a peptide according to thepresent invention), the method comprising: immunizing a geneticallyengineered non-human mammal comprising cells expressing said human majorhistocompatibility complex (MHC) class I or II with a soluble form of aMHC class I or II molecule being complexed with said HLA-restrictedantigen; isolating mRNA molecules from antibody producing cells of saidnon-human mammal; producing a phage display library displaying proteinmolecules encoded by said mRNA molecules; and isolating at least onephage from said phage display library, said at least one phagedisplaying said antibody specifically binding to said human majorhistocompatibility complex (MHC) class I or II being complexed with saidHLA-restricted antigen.

It is thus a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen, wherein theantibody preferably is a polyclonal antibody, monoclonal antibody,bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain classI major histocompatibility complexes, as well as other tools for theproduction of these antibodies are disclosed in WO 03/068201, WO2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen etal., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for thepurposes of the present invention are all explicitly incorporated byreference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isalso regarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 279, ora variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 279 or a variant thereof thatinduces T cells cross-reacting with said peptide, wherein said peptideis not the underlying full-length polypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:279 or a variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 279, wherein said peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 279.

The present invention further relates to the peptides according to theinvention, wherein the peptide is (chemically) modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or wherein the peptide is fusedto (or into) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe complete (full) human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine, in particular in the treatment of chronic lymphocyticleukemia, chronic myeloid leukemia and acute myeloid leukemia.

The present invention further relates to a host cell comprising anucleic acid according to the invention or an expression vectoraccording to the invention.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell, and preferably adendritic cell.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom said host cell or its culture medium.

The present invention further relates to the method according to thepresent invention, where-in the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID NO: 1 to SEQID NO: 279 or said variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellsselectively recognizes a cell which aberrantly expresses a polypeptidecomprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament. The present invention further relates to a useaccording to the present invention, wherein the medicament is activeagainst cancer.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine. The present inventionfurther relates to a use according to the invention, wherein themedicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are chronic lymphocytic leukemia,chronic myeloid leukemia and acute myeloid leukemia cells or other solidor hematological tumor cells such as other lymphoid neoplasms, forexample, Non-Hodgkin lymphoma, post-transplant lymphoproliferativedisorders (PTLD) as well as other myeloid neoplasms, such as primarymyelofibrosis, essential thrombocytopenia, polycythemia vera, as well asother neoplasms such as hepatocellular carcinoma, colorectal carcinoma,glioblastoma, gastric cancer, esophageal cancer, non-small cell lungcancer, small cell lung cancer, pancreatic cancer, renal cell carcinoma,prostate cancer, melanoma, breast cancer, gallbladder cancer andcholangiocarcinoma, urinary bladder cancer, uterine cancer, head andneck squamous cell carcinoma, mesothelioma.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of chronic lymphocytic leukemia, chronic myeloid leukemia andacute myeloid leukemia. The present invention also relates to the use ofthese novel targets for cancer treatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of a chronic lymphocytic leukemia,chronic myeloid leukemia and acute myeloid leukemia marker(poly)peptide, delivery of a toxin to a chronic lymphocytic leukemia,chronic myeloid leukemia and acute myeloid leukemia cell expressing acancer marker gene at an increased level, and/or inhibiting the activityof a chronic lymphocytic leukemia, chronic myeloid leukemia and acutemyeloid leukemia marker polypeptide) according to the invention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length chronic lymphocytic leukemia, chronic myeloidleukemia and acute myeloid leukemia marker polypeptides or fragmentsthereof may be used to generate the antibodies of the invention. Apolypeptide to be used for generating an antibody of the invention maybe partially or fully purified from a natural source, or may be producedusing recombinant DNA techniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 279polypeptide, or a variant or fragment thereof, can be expressed inprokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast,insect, or mammalian cells), after which the recombinant protein can bepurified and used to generate a monoclonal or polyclonal antibodypreparation that specifically bind the chronic lymphocytic leukemia,chronic myeloid leukemia and acute myeloid leukemia marker polypeptideused to generate the antibody according to the invention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). Forexample, the antibodies may be tested in ELISA assays or, Western blots,immunohistochemically staining of formalin-fixed cancers or frozentissue sections. After their initial in vitro characterization,antibodies intended for therapeutic or in vivo diagnostic use are testedaccording to known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (US 4,816,567), whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (pg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating chroniclymphocytic leukemia, chronic myeloid leukemia and acute myeloidleukemia, the efficacy of the therapeutic antibody can be assessed invarious ways well known to the skilled practitioner. For instance, thesize, number, and/or distribution of cancer in a subject receivingtreatment may be monitored using standard tumor imaging techniques. Atherapeutically-administered antibody that arrests tumor growth, resultsin tumor shrinkage, and/or prevents the development of new tumors,compared to the disease course that would occurs in the absence ofantibody administration, is an efficacious antibody for treatment ofcancer.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g. by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), and domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. Moreover, it could be expressed inT cells used for adoptive transfer. Further information can be found inWO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs isdescribed in WO 2012/056407A1. Further methods for the production aredisclosed in WO 2013/057586A1.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than1×10μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville,Maryland 20852, USA under Catalogue No CRL 1992; the Drosophila cellline Schneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Ljunggren et al.(Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T cellsare CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 279, or a variant aminoacid sequence thereof.

A number of other methods may be used for generating T cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL. Plebanski et al. (Plebanski et al., 1995) madeuse of autologous peripheral blood lymphocytes (PLBs) in the preparationof T cells. Furthermore, the production of autologous T cells by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus is possible. Also, B cells can be used in theproduction of autologous T cells. In addition, macrophages pulsed withpeptide or polypeptide, or infected with recombinant virus, may be usedin the preparation of autologous T cells. S. Walter et al. (Walter etal., 2003) describe the in vitro priming of T cells by using artificialantigen presenting cells (aAPCs), which is also a suitable way forgenerating T cells against the peptide of choice. In the presentinvention, aAPCs were generated by the coupling of preformed MHC:peptidecomplexes to the surface of polystyrene particles (microbeads) bybiotin:streptavidin biochemistry. This system permits the exact controlof the MHC density on aAPCs, which allows to selectively elicit high- orlow-avidity antigen-specific T cell responses with high efficiency fromblood samples. Apart from MHC:peptide complexes, aAPCs should carryother proteins with co-stimulatory activity like anti-CD28 antibodiescoupled to their surface. Furthermore, such aAPC-based systems oftenrequire the addition of appropriate soluble factors, e. g. cytokines,like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, andvaccinia-infected target cells. In addition, plant viruses may be used(see, for example, Porta et al. (Porta et al., 1994) which describes thedevelopment of cowpea mosaic virus as a high-yielding system for thepresentation of foreign peptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO 279.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for, anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to levels of expression in normal tissues orthat the gene is silent in the tissue from which the tumor is derivedbut in the tumor, it is expressed. By “over-expressed” the inventorsmean that the polypeptide is present at a level at least 1.2-fold ofthat present in normal tissue; preferably at least 2-fold, and morepreferably at least 5-fold or 10-fold the level present in normaltissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art. Reviews can be found in: Gattioni et al. and Morgan et al.(Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T-cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T cell. Thisengineered T cell can then be transferred to a patient for therapy ofcancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T cell, T-cell receptor or thenucleic acid encoding it, is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore, anymolecule of the present invention may be used as medicament or in themanufacture of a medicament. The molecule may be used by itself orcombined with other molecule(s) of the invention or (a) knownmolecule(s).

The present invention is further directed at a kit comprising: (a) acontainer containing a pharmaceutical composition as described above, insolution or in lyophilized form; (b) optionally a second containercontaining a diluent or reconstituting solution for the lyophilizedformulation; and (c) optionally, instructions for (i) use of thesolution or (ii) reconstitution and/or use of the lyophilizedformulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from chroniclymphocytic leukemia, chronic myeloid leukemia and acute myeloidleukemia, the medicament of the invention is preferably used to treatchronic lymphocytic leukemia, chronic myeloid leukemia and acute myeloidleukemia.

The present invention further relates to a method for producing apersonalized pharmaceutical for an individual patient comprisingmanufacturing a pharmaceutical composition comprising at least onepeptide selected from a warehouse of pre-screened TUMAPs, wherein the atleast one peptide used in the pharmaceutical composition is selected forsuitability in the individual patient. In one embodiment, thepharmaceutical composition is a vaccine. The method could also beadapted to produce T cell clones for down-stream applications, such asTCR isolations, or soluble antibodies, and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

As used herein, the term “warehouse” shall refer to a group or set ofpeptides that have been pre-screened for immunogenicity and/orover-presentation in a particular tumor type. The term “warehouse” isnot intended to imply that the particular peptides included in thevaccine have been pre-manufactured and stored in a physical facility,although that possibility is contemplated. It is expressly contemplatedthat the peptides may be manufactured de novo for each individualizedvaccine produced, or may be pre-manufactured and stored. The warehouse(e.g. in the form of a database) is composed of tumor-associatedpeptides which were highly overexpressed in the tumor tissue of chroniclymphocytic leukemia, chronic myeloid leukemia and acute myeloidleukemia patients with various HLA-A HLA-B and HLA-C alleles. It maycontain MHC class I and MHC class II peptides or elongated MHC class Ipeptides. In addition to the tumor associated peptides collected fromseveral chronic lymphocytic leukemia, chronic myeloid leukemia and acutemyeloid leukemia tissues, the warehouse may contain HLA-A*02, HLA-A*01,HLA-A*03, HLA-A*24, HLA-B*07, HLA-B*08 and HLA-B*44 marker peptides.These peptides allow comparison of the magnitude of T-cell immunityinduced by TUMAPS in a quantitative manner and hence allow importantconclusion to be drawn on the capacity of the vaccine to elicitanti-tumor responses. Secondly, they function as important positivecontrol peptides derived from a “non-self” antigen in the case that anyvaccine-induced T-cell responses to TUMAPs derived from “self” antigensin a patient are not observed. And thirdly, it may allow conclusions tobe drawn, regarding the status of immunocompetence of the patient.

TUMAPs for the warehouse are identified by using an integratedfunctional genomics approach combining gene expression analysis, massspectrometry, and T-cell immunology (XPresident®). The approach assuresthat only TUMAPs truly present on a high percentage of tumors but not oronly minimally expressed on normal tissue, are chosen for furtheranalysis. For initial peptide selection, chronic lymphocytic leukemia,chronic myeloid leukemia and acute myeloid leukemia samples frompatients and blood from healthy donors were analyzed in a stepwiseapproach:

1. HLA ligands from the malignant material were identified by massspectrometry

2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis wasused to identify genes over-expressed in the malignant tissue (chroniclymphocytic leukemia, chronic myeloid leukemia and acute myeloidleukemia) compared with a range of normal organs and tissues

3. Identified HLA ligands were compared to gene expression data.Peptides over-presented or selectively presented on tumor tissue,preferably encoded by selectively expressed or over-expressed genes asdetected in step 2 were considered suitable TUMAP candidates for amulti-peptide vaccine.

4. Literature research was performed in order to identify additionalevidence supporting the relevance of the identified peptides as TUMAPs

5. The relevance of over-expression at the mRNA level was confirmed byredetection of selected TUMAPs from step 3 on tumor tissue and lack of(or infrequent) detection on healthy tissues.

6. In order to assess, whether an induction of in vivo T-cell responsesby the selected peptides may be feasible, in vitro immunogenicity assayswere performed using human T cells from healthy donors as well as fromchronic lymphocytic leukemia, chronic myeloid leukemia and acute myeloidleukemia patients.

In an aspect, the peptides are pre-screened for immunogenicity beforebeing included in the warehouse. By way of example, and not limitation,the immunogenicity of the peptides included in the warehouse isdetermined by a method comprising in vitro T-cell priming throughrepeated stimulations of CD8+ T cells from healthy donors withartificial antigen presenting cells loaded with peptide/MHC complexesand anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rareexpression profile. In contrast to multi-peptide cocktails with a fixedcomposition as currently developed, the warehouse allows a significantlyhigher matching of the actual expression of antigens in the tumor withthe vaccine. Selected single or combinations of several “off-the-shelf”peptides will be used for each patient in a multitarget approach. Intheory, an approach based on selection of e.g. 5 different antigenicpeptides from a library of 50 would already lead to approximately 17million possible drug product (DP) compositions.

In an aspect, the peptides are selected for inclusion in the vaccinebased on their suitability for the individual patient based on themethod according to the present invention as described herein, or asbelow.

The HLA phenotype, transcriptomic and peptidomic data is gathered fromthe patient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing “warehouse” andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patients' tumor and,where possible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by amethod comprising: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; (b) comparingthe peptides identified in (a) with a warehouse (database) of peptidesas described above; and (c) selecting at least one peptide from thewarehouse (database) that correlates with a tumor-associated peptideidentified in the patient. For example, the TUMAPs presented by thetumor sample are identified by: (a1) comparing expression data from thetumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. Preferably, the sequences of MHCligands are identified by eluting bound peptides from MHC moleculesisolated from the tumor sample, and sequencing the eluted ligands.Preferably, the tumor sample and the normal tissue are obtained from thesame patient.

In addition to, or as an alternative to, selecting peptides using awarehousing (database) model, TUMAPs may be identified in the patient denovo, and then included in the vaccine. As one example, candidate TUMAPsmay be identified in the patient by (a1) comparing expression data fromthe tumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. As another example, proteins may beidentified containing mutations that are unique to the tumor samplerelative to normal corresponding tissue from the individual patient, andTUMAPs can be identified that specifically target the mutation. Forexample, the genome of the tumor and of corresponding normal tissue canbe sequenced by whole genome sequencing: For discovery of non-synonymousmutations in the protein-coding regions of genes, genomic DNA and RNAare extracted from tumor tissues and normal non-mutated genomic germlineDNA is extracted from peripheral blood mononuclear cells (PBMCs). Theapplied NGS approach is confined to the re-sequencing of protein codingregions (exome re-sequencing). For this purpose, exonic DNA from humansamples is captured using vendor-supplied target enrichment kits,followed by sequencing with e.g. a HiSeq2000 (Illumina). Additionally,tumor mRNA is sequenced for direct quantification of gene expression andvalidation that mutated genes are expressed in the patients' tumors. Theresultant millions of sequence reads are processed through softwarealgorithms. The output list contains mutations and gene expression.Tumor-specific somatic mutations are determined by comparison with thePBMC-derived germline variations and prioritized. The de novo identifiedpeptides can then be tested for immunogenicity as described above forthe warehouse, and candidate TUMAPs possessing suitable immunogenicityare selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient by the method asdescribed above; (b) comparing the peptides identified in a) with awarehouse of peptides that have been prescreened for immunogenicity andoverpresentation in tumors as compared to corresponding normal tissue;(c) selecting at least one peptide from the warehouse that correlateswith a tumor-associated peptide identified in the patient; and (d)optionally, selecting at least one peptide identified de novo in (a)confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of ˜2.5 mg/ml per peptide. The mixed solution is thendiluted 1:3 with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution is filtered througha 0.22 pm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 pL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from chronic lymphocytic leukemia, chronic myeloidleukemia and acute myeloid leukemia cells and since it was determinedthat these peptides are not or at lower levels present in normaltissues, these peptides can be used to diagnose the presence of acancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for chronic lymphocytic leukemia, chronic myeloid leukemia andacute myeloid leukemia. Presence of groups of peptides can enableclassification or sub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance. Thus, presence of peptides shows that thismechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate response markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses againstpeptides can be considered in the assessment of side effects. Monitoringof lymphocyte responses might also be a valuable tool for follow-upexaminations of transplantation therapies, e.g. for the detection ofgraft versus host and host versus graft diseases.

The present invention will now be described in the following exampleswhich describe preferred embodiments thereof, and with reference to theaccompanying figures, nevertheless, without being limited thereto. Forthe purposes of the present invention, all references as cited hereinare incorporated by reference in their entireties.

FIGURES

FIGS. 1A through 1W show exemplary expression profile of source genes ofthe present invention that are over-expressed in different cancersamples. Tumor (black dots) and normal (grey dots) samples are groupedaccording to organ of origin, and box-and-whisker plots representmedian, 25th and 75th percentile (box), and minimum and maximum(whiskers) RPKM values. Normal organs are ordered according to riskcategories. RPKM=reads per kilobase per million mapped reads. Normalsamples: blood cells; blood vessel; brain; heart; liver; lung; adipose:adipose tissue; adren.gl.: adrenal gland; bile duct; bladder; BM: bonemarrow; cartilage; esoph: esophagus; eye; gallb: gallbladder; head andneck; kidney; large_int: large intestine; LN: lymph node; nerve;pancreas; parathyr: parathyroid; perit: peritoneum; pituit: pituitary;skel.mus: skeletal muscle; skin; small_int: small intestine; spleen;stomach; thyroid; trachea; ureter; breast; ovary; placenta; prostate;testis; thymus; uterus. Tumor samples: AML: acute myeloid leukemia; CLL:chronic lymphocytic leukemia; NHL: non-hodgkin lymphoma. FIG. 1A) Genesymbol: S100Z, Peptide: TMIRIFHRY (SEQ ID No.: 2), FIG. 1B) Gene symbol:PAX5, Peptide: YSHPQYSSY (SEQ ID No.: 9), 1C) Gene symbol: FLT3,Peptide: SLFEGIYTI (SEQ ID No.: 19), 1D) Gene symbol: RALGPS2, Peptide:ILHAQTLKI (SEQ ID No.: 22), 1E) Gene symbol: FCRL2, Peptide: KTSNIVKIK(SEQ ID No.: 32), 1F) Gene symbol: KBTBD8, Peptide: RSKEYIRKK (SEQ IDNo.: 40), 1G) Gene symbols: ZNF92, Peptide: KAFNQSSTLTK (SEQ ID No.:52), 1H) Gene symbol: ADAM28, Peptide: KYIEYYLVL (SEQ ID No.: 53), 11)Gene symbol: FLT3, Peptide: IFKEHNFSF (SEQ ID No.: 61), 1J) Gene symbol:ZNF92, Peptide: KAFSWSSAF (SEQ ID No.: 76), 1K) Gene symbol: FCRL3,Peptide: IPVSHPVL (SEQ ID No.: 85), 1L) Gene symbol: CDK6, Peptide:FGLARIYSF (SEQ ID No.: 110), 1M) Gene symbol: CLEC17A, Peptide:VTLIKYQEL (SEQ ID No.: 111), 1N) Gene symbol: RALGPS2, Peptide: YIKTAKKL(SEQ ID No.: 117), 10) Gene symbol: CDK6, Peptide: GEGAYGKVF (SEQ IDNo.: 129), 1P) Gene symbol: FCRL2, Peptide: RENQVLGSGW (SEQ ID No.:139), 1Q) Gene symbol: FLT3, Peptide: REYEYDLKWEF (SEQ ID No.: 141), 1R)Gene symbol: BMF, Peptide: VTEEPQRLFY (SEQ ID No.: 189), 1S) Genesymbol: FCER2, Peptide: LLWHWDTTQSLK (SEQ ID No.: 212), 1T) Gene symbol:CDK6, Peptide: MPLSTIREV (SEQ ID No.: 231), 1U) Gene symbol: CLEC17A,Peptide: SPRVYWLGL (SEQ ID No.: 233), 1V) Gene symbol: PMAIP1, Peptide:QPSPARAPAEL (SEQ ID No.: 247), 1W) Gene symbol: CDK6, Peptide:AEIGEGAYGKVF (SEQ ID No.: 260).

FIG. 2 shows exemplary results of peptide-specific in vitro CD8+ T cellresponses of a healthy HLA-A*02+ donor. CD8+ T cells were primed usingartificial APCs coated with anti-CD28 mAb and HLA-A*02 in complex withSeqID No 278 peptide (YLDRKLLTL, Seq ID NO: 278) (A, left panel). Afterthree cycles of stimulation, the detection of peptide-reactive cells wasperformed by 2D multimer staining with A*02/SeqID No 278 (A). Rightpanel (B) show control staining of cells stimulated with irrelevantA*02/peptide complexes. Viable singlet cells were gated for CD8+lymphocytes. Boolean gates helped excluding false-positive eventsdetected with multimers specific for different peptides. Frequencies ofspecific multimer+cells among CD8+ lymphocytes are indicated.

FIG. 3 shows exemplary results of peptide-specific in vitro CD8+ T cellresponses of a healthy HLA-A*24+ donor. CD8+ T cells were primed usingartificial APCs coated with anti-CD28 mAb and HLA-A*24 in complex withSeqID No 279 peptide (A, left panel). After three cycles of stimulation,the detection of peptide-reactive cells was performed by 2D multimerstaining with A*24/SeqID No 279 (LYIDRPLPYL, Seq ID NO: 279) (A). Rightpanel (B) shows control staining of cells stimulated with irrelevantA*24/peptide complexes. Viable singlet cells were gated for CD8+lymphocytes. Boolean gates helped excluding false-positive eventsdetected with multimers specific for different peptides. Frequencies ofspecific multimer+cells among CD8+ lymphocytes are indicated.

FIG. 4 shows exemplary results of peptide-specific in vitro CD8+ T cellresponses of a healthy HLA-A*01+ donor. CD8+ T cells were primed usingartificial APCs coated with anti-CD28 mAb and HLA-A*01 in complex withSeq ID NO: 12 peptide (ATDIVDSQY, Seq ID NO: 12; A, left panel) and SeqID NO: 192 peptide (RSDPGGGGLAY, Seq ID NO: 192; B, left panel),respectively. After three cycles of stimulation, the detection ofpeptide-reactive cells was performed by 2D multimer staining withA*01/Seq ID NO: 12 (A) or A*01/Seq ID NO: 192 (B). Right panels (A andB) show control staining of cells stimulated with irrelevantA*01/peptide complexes. Viable singlet cells were gated for CD8+lymphocytes. Boolean gates helped excluding false-positive eventsdetected with multimers specific for different peptides. Frequencies ofspecific multimer+cells among CD8+ lymphocytes are indicated.

FIG. 5 shows exemplary results of peptide-specific in vitro CD8+ T cellresponses of a healthy HLA-A*02+ donor. CD8+ T cells were primed usingartificial APCs coated with anti-CD28 mAb and HLA-A*02 in complex withSeq ID NO: 19 peptide (SLFEGIYTI, Seq ID NO: 19; A, left panel) and SeqID NO: 26 peptide (SLYVQQLKI, Seq ID NO: 26; B, left panel),respectively. After three cycles of stimulation, the detection ofpeptide-reactive cells was performed by 2D multimer staining withA*02/Seq ID NO: 19 (A) or A*02/Seq ID NO: 26 (B). Right panels (A and B)show control staining of cells stimulated with irrelevant A*02/peptidecomplexes. Viable singlet cells were gated for CD8+ lymphocytes. Booleangates helped excluding false-positive events detected with multimersspecific for different peptides. Frequencies of specific multimer+cellsamong CD8+ lymphocytes are indicated.

FIG. 6 shows exemplary results of peptide-specific in vitro CD8+ T cellresponses of a healthy HLA-A*03+ donor. CD8+ T cells were primed usingartificial APCs coated with anti-CD28 mAb and HLA-A*03 in complex withSeq ID NO: 45 peptide (VVFPFPVNK, Seq ID NO: 45; A, left panel) andSeqID No 215 peptide (KATGAATPK, Seq ID NO: 215; B, left panel),respectively. After three cycles of stimulation, the detection ofpeptide-reactive cells was performed by 2D multimer staining withA*03/Seq ID NO: 45 (A) or A*03/Seq ID NO: 215 (B). Right panels (A andB) show control staining of cells stimulated with irrelevantA*03/peptide complexes. Viable singlet cells were gated for CD8+lymphocytes. Boolean gates helped excluding false-positive eventsdetected with multimers specific for different peptides. Frequencies ofspecific multimer+cells among CD8+ lymphocytes are indicated.

FIG. 7 shows exemplary results of peptide-specific in vitro CD8+ T cellresponses of a healthy HLA-A*24+ donor. CD8+ T cells were primed usingartificial APCs coated with anti-CD28 mAb and HLA-A*24 in complex withSeq ID NO: 53 peptide (KYIEYYLVL, Seq ID NO: 53; A, left panel) and SeqID NO: 68 peptide (LYQDRFDYL, Seq ID NO: 68; B, left panel),respectively. After three cycles of stimulation, the detection ofpeptide-reactive cells was performed by 2D multimer staining withA*02/Seq ID NO: 53 (A) or A*24/Seq ID NO: 68 (B). Right panels (A and B)show control staining of cells stimulated with irrelevant A*24/peptidecomplexes. Viable singlet cells were gated for CD8+ lymphocytes. Booleangates helped excluding false-positive events detected with multimersspecific for different peptides. Frequencies of specific multimer+cellsamong CD8+ lymphocytes are indicated.

FIG. 8 shows exemplary results of peptide-specific in vitro CD8+ T cellresponses of a healthy HLA-B*07+ donor. CD8+ T cells were primed usingartificial APCs coated with anti-CD28 mAb and HLA-B*07 in complex withSeqID No 233 peptide (SPRVYWLGL, Seq ID NO: 233; A, left panel) and SeqID NO: 84 peptide (SPKLQIAAM, Seq ID NO: 84; B, left panel),respectively. After three cycles of stimulation, the detection ofpeptide-reactive cells was performed by 2D multimer staining withB*07/Seq ID NO: 233 (A) or B*07/Seq ID NO: 84 (B). Right panels (A andB) show control staining of cells stimulated with irrelevantB*07/peptide complexes. Viable singlet cells were gated for CD8+lymphocytes. Boolean gates helped excluding false-positive eventsdetected with multimers specific for different peptides. Frequencies ofspecific multimer+cells among CD8+ lymphocytes are indicated.

FIG. 9 shows exemplary results of peptide-specific in vitro CD8+ T cellresponses of a healthy HLA-B*44+ donor. CD8+ T cells were primed usingartificial APCs coated with anti-CD28 mAb and HLA-B*44 in complex withSEQ ID NO: 145 peptide (AEPLVGQRW, SEQ ID NO: 145; A, left panel) andSEQ ID NO: 171 peptide (SEDLAVHLY, SEQ ID NO: 171; B, left panel),respectively. After three cycles of stimulation, the detection ofpeptide-reactive cells was performed by 2D multimer staining withB*44/SEQ ID NO: 145 (A) or B*44/SEQ ID NO: 171 (B). Right panels (A andB) show control staining of cells stimulated with irrelevantB*44/peptide complexes. Viable singlet cells were gated for CD8+lymphocytes. Boolean gates helped excluding false-positive eventsdetected with multimers specific for different peptides. Frequencies ofspecific multimer+cells among CD8+ lymphocytes are indicated.

EXAMPLES Example 1

IDentification of Tumor Associated Peptides Presented on the CellSurface

Tissue Samples

Patients' tumor samples and normal tissues were obtained from theUniversity Hospital Tübingen (Tübingen, Germany). Written informedconsents of all patients had been given before blood draw. PBMC wereisolated from blood samples using Ficoll-Hypaque density gradientcentrifugation immediately after blood draw. PBMC pellets wereshock-frozen immediately after purification and stored until isolationof TUMAPs at -70° C. or below.

Isolation of HLA Peptides from Tissue Samples HLA peptide pools fromshock-frozen samples were obtained by immune precipitation according toa slightly modified protocol (Falk et al., 1991; Seeger et al., 1999)using the HLA-A*02-specific antibody BB7.2, the HLA-A, -B, C-specificantibody W6/32, CNBr-activated sepharose, acid treatment, andultrafiltration.

Mass Spectrometry Analyses

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (Ultimate 3000 RSLC NanoUHPLC System, Dionex) and the eluting peptides were analyzed inLTQ-Orbitrap and Fusion Lumos hybrid mass spectrometers (ThermoElectron)equipped with an ESI source. Peptide samples were loaded with 3% ofsolvent B (20% H₂O, 80% acetonitrile and 0.04% formic acid) on a 2 cmPepMap 100 C18 Nanotrap column (Dionex) at a flowrate of 4 pl/min for 10min. Separation was performed on either 25 cm or 50 cm PepMap C18columns with a particle size of 2 pm (Dionex) mounted in a column ovenrunning at 50° C. The applied gradient ranged from 3 to 40% solvent Bwithin 90 min at a flow rate of 300 nl/min (for 25 cm columns) or 140min at a flow rate of 175 nl/min (for 50 cm columns). (Solvent A: 99%H₂O, 1% ACN and 0.1% formic acid; Solvent B: 20% H₂O, 80% ACN and 0.1%formic acid).

Mass spectrometry analysis was performed in data dependent acquisitionmode employing a top five method (i.e. during each survey scan the fivemost abundant precursor ions were selected for fragmentation).Alternatively, a TopSpeed method was employed for analysis on FusionLumos instruments.

Survey scans were recorded in the Orbitrap at a resolution of 60,000(for Orbitrap XL) or 120,000 (for Orbitrap Fusion Lumos). MS/MS analysiswas performed by collision induced dissociation (CID, normalizedcollision energy 35%, activation time 30 ms, isolation width 1.3 m/z)with subsequent analysis in the linear trap quadrupole (LTQ). Mass rangefor HLA class I ligands was limited to 400-650 m/z with charge states 2+and 3+ selected for fragmentation. For HLA class II mass range was setto 300-1500 m/z allowing for fragmentation for all positive chargestates 2.

Tandem mass spectra were interpreted by MASCOT or SEQUEST databasesearch at a fixed Percolator false discovery rate (q≤0.05) andadditional manual control. In cases where the identified peptidesequence was uncertain it was additionally validated by comparison ofthe generated natural peptide fragmentation pattern with thefragmentation pattern of a synthetic sequence-identical referencepeptide.

Table 8a and 8b show the presentation on various cancer entities forselected peptides, and thus the particular relevance of the peptides asmentioned for the diagnosis and/or treatment of the cancers as indicated(e.g. peptide SEQ ID No. 3 for AML, CML (Table 8a) and for GBC, MEL,NHL, NSCLC and UBC (Table 8b), peptide SEQ ID No. 4 for CLL (Table 8a)and for BRCA, CCC, CRC, GBC, GC, GEJC, HCC, HNSCC, MEL, NSCLCadeno,NSCLCother, NSCLCsquam, OSCAR, PACA, PRCA, SCLC, UBC, UEC (Table 8b)).

TABLE 8a Overview of presentation of selected tumor-associated peptides of the present invention across entities (diseases).CLL = chronic lymphocytic leukemia, AML =acute myeloid leukemia, CML = Chronic  myeloid leukemia. SEQ IDPeptide Presentation No. Sequence on cancer entities   1 LTEGHSGNYY CLL  2 TMIRIFHRY AML   3 YINPAKLTPY CLL   4 ALDQNKMHY AML   5 GTDVLSTRYAML, CML   6 VTEGVAQTSFY CLL   7 FMDSESFYY CLL   8 STDSAGSSY CLL   9YSHPQYSSY CLL  10 YSDIGHLL CLL  11 AAADHHSLY AML  12 ATDIVDSQY CLL  13ITDIHIKY CLL  14 TFDLTVVSY CLL  15 SVADIRNAY CLL  16 WIGDKSFEY AML  17KAYNRVIFV CLL  18 YLLPSVVLL CLL  19 SLFEGIYTI AML  20 FSLEDLVRI CLL  21FLFDKLLLI CLL  22 ILHAQTLKI CLL  23 FAFSGVLRA AML  24 KLGPVAVSI CLL  25YLNEKSLQL AML, CML  26 SLYVQQLKI CLL  27 RLIAKEMNI CLL  28 VILESIFLK CLL 29 RIYDEILQSK AML  30 RTYGFVLTF CLL  31 ATFNKLVSY AML  32 KTSNIVKIK CLL 33 SVFEGDSIVLK CLL  34 SVYSETSNMDK CLL  35 ATKSPAKPK CLL  36 KAKAAAKPKCLL  37 KAKKPAGAAK CLL  38 KARKSAGAAK CLL  39 IVIQLRAQK CLL  40RSKEYIRKK CLL  41 SVAHLLSKY CLL  42 SVSSSTHFTR CLL  43 KLMETSMGF CLL  44KVYDPVSEY CLL  45 VVFPFPVNK AML, CML  46 RVFPSPMRI CLL  47 SVLDLSVHK CLL 48 RIKPPGPTAVPK AML  49 GLLEEALFY AML  50 GVFNTLISY AML  51 ASTTVLALKCLL  52 KAFNQSSTLTK CLL  53 KYIEYYLVL CLL  54 QQALNFTRF CLL  55IFVARLYYF CLL  56 KYSSGFRNI CLL  57 RFPPTPPLF CLL  58 KYLADLPTL CLL  59GLYEGTGRLF AML  60 TQDPHVNAFF CLL  61 IFKEHNFSF AML  62 YYLSHLERI AML 63 IYFSNTHFF AML  64 SFQSKATVF AML  65 AYLKQVLLF CLL  66 SQPAVATSF AML 67 VFLPSEGFNF AML  68 LYQDRFDYL AML  69 EYNTIKDKF CLL  70 LYSDIGHLL CLL 71 RYLGKNWSF AML  72 TYVENLRLL CLL  73 TYPQLEGFKF CLL  74 SYADNILSF CLL 75 RFYLLTEHF AML  76 KAFSWSSAF CLL  77 RPNGNSLFTSA CLL  78 RPRGLALVLAML, CML  79 SPVPSHWMVA CLL  80 KPLFKVSTF CLL  81 SESPWLHAPSL CLL  82APFGFLGMQSL CLL  83 IPVSRPIL CLL  84 SPKLQIAAM CLL  85 IPVSHPVL CLL  86IPASHPVL CLL  87 FPAPILRAV CLL  88 MPDPHLYHQM CLL  89 FPETVNNLL CLL  90KPKAAKPKA CLL  91 KPKAAKPKAA CLL  92 KAKKPAGAA CLL  93 KARKSAGAA CLL  94KPKAAKPKKAAA CLL  95 KPKAAKPKTA CLL  96 KPKKAPKSPA CLL  97 LPFGKIPILAML, CML  98 YPIALTRAEM CLL  99 SPRAINNLVL CLL 100 YPYQERVFL CML 101NPRYPNYMF CLL 102 LPLSMEAKI CML 103 IPANTEKASF CLL 104 RPMTPTQIGPSL CLL105 NPLTKLLAI AML 106 KAFKWFSAL CLL 107 QAAQRTAL CLL 108 ILAIRQNAL CLL109 LGHVRYVL CLL 110 FGLARIYSF AML, CML 111 VTLIKYQEL CLL 112 APLLRHWELCLL 113 DANSRTSQL CLL 114 HNALRILTF AML 115 ELYQRIYAF AML 116 TLKIRAEVLCLL 117 YIKTAKKL CLL 118 FEKEKKESL CLL 119 DLRTKEVVF CLL 120 VPPKKHLLCLL 121 RPKKVNTL CLL 122 KELPGVKKY CLL 123 EENPGKFLF CLL 124 SESLPKEAFCLL 125 SESTFDRTF CLL 126 EENKPGIVY CLL 127 TEYPVFVY AML 128 GENDRLNHTYCLL 129 GEGAYGKVF AML 130 EEEHGKGREY CLL 131 EEFETIERF CLL 132 GELPAVRDLCLL 133 AEHNFVAKA CLL 134 SEYADTHYF CLL 135 NEIKVYITF AML, CML 136AEYKGRVTL CLL 137 GELGGSVTI CLL 138 SQAPAARAF CLL 139 RENQVLGSGW CLL 140EYDLKWEF AML 141 REYEYDLKWEF AML 142 TEIFKEHNF AML 143 YEYDLKWEF AML 144TEGKRYFTW CLL 145 AEPLVGQRW CLL 146 SESKTVVTY CLL 147 KEVPRSYEL CLL 148REYNEYENI CLL 149 SEKETVAYF CLL 150 EEVTDRSQL CLL 151 EVDASIFKAW CLL 152AELLAKELY CLL 153 KEFEQVPGHL CLL 154 AEPGPVITW CLL 155 NEFPVIVRL CLL 156FEVESLFQKY CLL 157 VEIAEAIQL CLL 158 GENEDNRIGL CLL 159 GELLGRQSF CLL160 EEETILHFF CLL 161 EEGDTLLHLF CLL 162 DEAQARAAF AML 163 EEVVMGLLEYAML 164 SEYSHLTRV AML 165 VELDLQRSV AML 166 NEVLASKY CLL 167 KEIGAAVQALCLL 168 QEIQSLLTNW CLL 169 EENGEVKEL CLL 170 SENEQRRMF CLL 171 SEDLAVHLYCLL 172 VEDGLFHEF CLL 173 KEYDFGTQL AML 174 TDKSFPNAY CLL 175 HEIDGKALFLAML 176 AENAVSNLSF CLL 177 QENMQIQSF CLL 178 REYEHYVVTEL CLL 179AEIKQTEEKY AML 180 EEPAFNVSY CLL 181 GEIKEPLEI CLL 182 AQNLSIIQY CLL 183GESQDSTTAL CLL 184 RMPPFTQAF CLL 185 SEGDNVESW CLL 186 NEQKIVRF CLL 187SDAQRPSSF CLL 188 YVDAGTPMY CLL 189 VTEEPQRLFY CLL 190 HVDQDLTTY AML 191ISEAGKDLLY AML, CML, CLL 192 RSDPGGGGLAY AML 193 LTDSEKGNSY CLL 194YTDKKSIIY CLL 195 YSDKEFAGSY CLL 196 FTDIDGQVY CLL 197 SLADVHIEV CLL 198KLLGYDVHV AML 199 AMPDSPAEV AML 200 VMLQINPKL CLL 201 ILAAVETRL CLL 202MVALPMVLV CLL 203 FLLPKVQSI CLL 204 FLLPKVQSIQL CLL 205 FLINTNSEL CLL206 SLMDLQERL CLL 207 KLSDNILKL CLL 208 KLNPQQAPLY CLL 209 KTLPAMLGTGKCLL 210 RMYSQLKTLQK CLL 211 ATYNKQPMYR CML 212 LLWHWDTTQSLK CLL 213RVYNIYIRR AML 214 ATGAATPKK CLL 215 KATGAATPK CLL 216 RIKAPSRNTIQK CLL217 TTVPHVFSK CLL 218 RVLTGVFTK CLL 219 HSYSSPSTK CLL 220 SISNLVFTY AML221 LLNRHILAH CLL 222 RYLDEINLL AML 223 RRMYPPPLI CLL 224 VYEYVVERF CLL225 LPARFYQAL CLL 226 YLNRHLHTW CLL 227 APINKAGSFL CLL 228 SPRITFPSL CLL229 SPLGSLARSSL CLL 230 KPMKSVLVV CLL 231 MPLSTIREV AML, CML 232APRPAGSYL CLL 233 SPRVYWLGL CLL 234 SPKESENAL CLL 235 SPSLPSRTL CLL 236RPSNKAPLL CLL 237 SPWLHAPSL CLL 238 SPRSWIQVQI CLL 239 APSKTSLIM CLL 240SPSLPNITL CLL 241 APAPAEKTPV CLL 242 SPFSFHHVL CLL 243 LPKVQSIQL CLL 244MPSSDTTVTF CLL 245 SPLSHHSQL CLL 246 YPGWHSTTI AML 247 QPSPARAPAEL CLL248 LPYDSKHQI CLL 249 SPADHRGYASL AML 250 VPNLQTVSV CLL 251 QPRLFTMDLCLL 252 RPHIPISKL CLL 253 RPFADLLGTAF CLL 254 SPRNLQPQRAAL CLL 255YPGSDRIML CLL 256 SPYKKLKEAL CLL 257 KEFFFVKVF CLL 258 EELFRDGVNW CLL259 EENTLVQNY CLL 260 AEIGEGAYGKVF AML 261 NEIEHIPVW CLL 262 QENQAETHAWCLL 263 REAGFQVKAY CLL 264 SEDHSGSYW CLL 265 QEVDASIFKAW CLL 266VDASIFKAW CLL 267 KEKFPINGW CLL 268 NEDKGTKAW CLL 269 KELEDLNKW CLL 270AESEDLAVHL CLL 271 AESEDLAVHLY CLL 272 KEFELRSSW CLL 273 AEIEIVKEEF CLL274 GEAVTDHPDRLW CLL 275 TENPLTKLL AML 276 EEEGNLLRSW CLL 277 EEGNLLRSWCLL

TABLE 8b Overview of presentation of selected tumor-associated peptides of the present invention across entities (diseases).BRCA = breast cancer, CCC = bile duct cancer,GBM = brain cancer, CRC = colorectal carcinoma,OSCAR = esophageal cancer, GBC = gallbladderadenocarcinoma, GC = gastric cancer, HNSCC =head and neck squamous cell carcinoma, HCC =hepatocellular carcinoma, MEL = melanoma, NHL =non-Hodgkin lymphoma, NSCLCadeno = non-smallcell lung cancer adenocarcinoma, NSCLCother =NSCLC samples that could not unambiguously beassigned to NSCLCadeno or NSCLCsquam,NSCLCsquam = squamous cell non-small cell lungcancer, OC = ovarian cancer, PACA = pancreaticcancer, PRCA = prostate cancer and benignprostate hyperplasia, RCC = renal cell car-cinoma, SCLC = small cell lung cancer, UBC =urinary bladder cancer, UEC = uterine cancer. Seq IDPeptide Presentation No Sequence on tumor types   1 LTEGHSGNY NHL Y   3YINPAKLTPY GBC, MEL, NHL, NSCLCother, UBC   4 ALDQNKMHYBRCA, CCC, CRC, GBC, GC, GEJC, HCC, HNSCC, MEL, NSCLCadeno,NSCLCother, NSCLCsquam, OSCAR, PACA, PRCA, SCLC, UBC, UEC   5 GTDVLSTRYBRCA, CRC, GBC, GC, GEJC, MEL, NHL, NSCLCadeno, NSCLCother,NSCLCsquam, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC   6 VTEGVAQTSFBRCA, CCC, GBC, GBM, GC, GEJC, Y HCC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA,PRCA, RCC, SCLC, UEC   7 FMDSESFYY BRCA, GBC, GC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCsquam   8 STDSAGSSY NHL  10 YSDIGHLLBRCA, CCC, GC, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC,PACA, RCC, UEC  11 AAADHHSLY GC, SCLC  13 ITDIHIKYBRCA, CRC, GBC, GBM, GC, GEJC, HNSCC, MEL, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OSCAR, PACA, PRCA, RCC, SCLC, UBC  14 TFDLTVVSYBRCA, CCC, CRC, GBC, GBM, GC, HCC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA,PRCA, RCC, SCLC, UBC, UEC  15 SVADIRNAY BRCA, CCC, CRC, GBC, GBM, GC,HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother,NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC  16 WIGDKSFEYBRCA, CRC, GBC, GBM, GC, HCC, MEL, NSCLCadeno, NSCLCsquam,OSCAR, PACA, PRCA, RCC  17 KAYNRVIFV BRCA, CCC, CRC, GBC, GBM, GEJC,HCC, HNSCC, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC,OSCAR, PACA, SCLC, UBC, UEC  18 YLLPSVVLL BRCA, CCC, CRC, GBC, GBM, GC,GEJC, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother,NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC  20 FSLEDLVRI NHL 21 FLFDKLLLI BRCA, CCC, CRC, GBC, GBM, GC, GEJC, HCC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA,PRCA, RCC, SCLC, UBC, UEC  22 ILHAQTLKI HCC, NHL, NSCLCsquam, PRCA  23FAFSGVLRA BRCA, CRC, GBC, GEJC, HCC, HNSCC, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OC, OSCAR, PRCA, SCLC, UBC, UEC  24 KLGPVAVSIHCC, HNSCC, NHL, NSCLCsquam, OSCAR, PRCA  25 YLNEKSLQLBRCA, CCC, CRC, GEJC, HCC, MEL, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OC, PRCA, RCC, SCLC, UBC, UEC  26 SLYVQQLKIHNSCC, NHL, OC, PRCA  27 RLIAKEMNI HCC, NHL  28 VILESIFLKGBC, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PRCA, UEC  29RIYDEILQSK RCC  31 ATFNKLVSY CRC, SCLC, UBC  33 SVFEGDSIVLNHL, NSCLCadeno, NSCLCother, OC K  34 SVYSETSNMBRCA, MEL, NHL, NSCLCsquam, DK OC, OSCAR, RCC, UEC  41 SVAHLLSKY UEC  42SVSSSTHFTR GC, HNSCC, NHL, NSCLCadeno, NSCLCsquam, UEC  43 KLMETSMGFBRCA, CRC, GBC, GBM, GC, HCC, HNSCC, MEL, NHL, NSCLCother,NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC  44 KVYDPVSEYBRCA, CCC, GBC, GBM, HCC, MEL, NHL, NSCLCadeno, NSCLCother,NSCLCsquam, OC, OSCAR, PACA, RCC, SCLC, UBC, UEC  45 VVFPFPVNKGBM, NHL, NSCLCother, OC, SCLC, UEC  46 RVFPSPMRIMEL, NHL, NSCLCsquam, OSCAR, SCLC  47 SVLDLSVHKCCC, HCC, NHL, OSCAR, PRCA, SCLC, UEC  48 RIKPPGPTAV BRCA, MEL PK  49GLLEEALFY BRCA, CRC, GBC, GC, HCC, MEL, NSCLCadeno, NSCLCsquam, OSCAR,PACA, PRCA, RCC  50 GVFNTLISY MEL, PRCA  51 ASTTVLALK NHL  53 KYIEYYLVLGC, NSCLCadeno, NSCLCsquam, UEC  54 QQALNFTRF GBC, MEL, NHL, NSCLCadeno,NSCLCsquam, OC, RCC, SCLC  55 IFVARLYYF BRCA, CRC, GBC, GC, HCC, MEL,NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA,PRCA, RCC, SCLC, UBC, UEC  56 KYSSGFRNI BRCA, CCC, CRC, GBC, GBM, GC,HCC, MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC,OSCAR, PACA, PRCA, RCC, SCLC, UEC  57 RFPPTPPLFHNSCC, NHL, NSCLCsquam, OSCAR, UEC  58 KYLADLPTLBRCA, CCC, CRC, GBC, GBM, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OC, OSCAR, PRCA, RCC, SCLC, UEC  60 TQDPHVNAFFBRCA, CRC, GBC, GEJC, HCC, HNSCC, NHL, NSCLCadeno, NSCLCother,NSCLCsquam, OC, OSCAR, PACA, PRCA, SCLC, UBC, UEC  62 YYLSHLERIGBC, HNSCC, NSCLCadeno, NSCLCother, NSCLCsquam, UBC  65 AYLKQVLLFBRCA, CCC, CRC, GBC, GBM, GC, HCC, MEL, NHL, NSCLCadeno, NSCLCother,NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC  66 SQPAVATSFCRC, SCLC  67 VFLPSEGFNF MEL, NSCLCadeno, SCLC  68 LYQDRFDYLGC, MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, PRCA, SCLC, UEC  69EYNTIKDKF GBC, GC, HCC, MEL, NHL, NSCLCadeno, NSCLCother,NSCLCsquam, OSCAR, PACA, SCLC, UEC  70 LYSDIGHLLCRC, GBC, GBM, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother,NSCLCsquam, OSCAR, PRCA, RCC, UBC, UEC  71 RYLGKNWSF GBC, OC, RCC  72TYVENLRLL BRCA, CCC, CRC, GBC, GBM, GC,HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC,OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC  73 TYPQLEGFKFBRCA, CRC, GBC, GC, GEJC, HCC, MEL, NHL, NSCLCadeno, NSCLCother,NSCLCsquam, OC, OSCAR, PACA, PRCA, SCLC, UBC, UEC  74 SYADNILSFBRCA, CRC, GBC, GBM, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC  75RFYLLTEHF CRC, GBC, HCC, NHL, NSCLCadeno, NSCLCsquam, OSCAR, SCLC  76KAFSWSSAF BRCA, GBM, NHL, NSCLCsquam, PRCA, SCLC, UBC  77 RPNGNSLFTSHCC, MEL, NHL, PRCA, SCLC, UEC A  78 RPRGLALVLBRCA, CCC, CRC, GBC, GBM, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC  79SPVPSHVVMV CCC, MEL, NHL, PRCA, SCLC, UBC A  80 KPLFKVSTFNHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, RCC, UEC  84SPKLQIAAM NHL, OC  85 IPVSHPVL NHL  86 IPASHPVLGBC, MEL, NHL, NSCLCsquam, OSCAR, PACA, SCLC  87 FPAPILRAVGBC, GC, MEL, NHL, NSCLCother, UBC  88 MPDPHLYHQ MEL, NHL, NSCLCsquam M 89 FPETVNNLL NSCLCadeno, NSCLCother, NSCLCsquam, PACA  97 LPFGKIPILBRCA, CRC, GBC, GBM, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC  98YPIALTRAEM BRCA, CCC, GBC, GC, HNSCC, MEL, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, SCLC, UEC  99SPRAINNLVL NHL 100 YPYQERVFL NHL, NSCLCadeno, NSCLCsquam 101 NPRYPNYMFNHL 104 RPMTPTQIGP NHL SL 105 NPLTKLLAI CCC, MEL, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OC 106 KAFKWFSAL NHL 108 ILAIRQNAL CCC, NHL, OC109 LGHVRYVL CCC, GBM, NHL, UBC 110 FGLARIYSF BRCA, CCC, GBC, GC, HCC,HNSCC, MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC,OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 112 APLLRHWELBRCA, CCC, CRC, GBC, HCC, HNSCC, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OC, OSCAR, PACA, SCLC 113 DANSRTSQLMEL, NHL, NSCLCadeno, NSCLCother, UEC 114 HNALRILTFBRCA, NHL, NSCLCother, NSCLCsquam 115 ELYQRIYAF HNSCC, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, PRCA, SCLC 116 TLKIRAEVL CCC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PRCA, SCLC 118 FEKEKKESLOC 119 DLRTKEVVF GC, NHL 122 KELPGVKKY CCC, CRC, GC, HCC, NHL,NSCLCadeno, NSCLCsquam, OSCAR, PACA, RCC, UEC 123 EENPGKFLFHNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam, OSCAR, PRCA 124 SESLPKEAFBRCA, CCC, CRC, GBC, GBM, GC, GEJC, HCC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA,PRCA, RCC, SCLC, UBC, UEC 126 EENKPGIVY CRC, HCC, NHL 128 GENDRLNHT NHLY 129 GEGAYGKVF HCC, HNSCC, NSCLCadeno, OC, OSCAR, RCC 131 EEFETIERFBRCA, CCC, CRC, GBC, GBM, GC, GEJC, HCC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA,PRCA, RCC, SCLC, UBC, UEC 132 GELPAVRDL CRC, GBC, GBM, GC, HCC, HNSCC,NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA 133 AEHNFVAKABRCA, GBM, HNSCC, MEL, NHL, NSCLCadeno, OC, OSCAR 134 SEYADTHYF NHL 135NEIKVYITF BRCA, CRC, HNSCC, NSCLCadeno, NSCLCsquam, PRCA, RCC 136AEYKGRVTL NHL, OSCAR 138 SQAPAARAF NHL, NSCLCadeno 139 RENQVLGSG NHL W143 YEYDLKWEF NHL 144 TEGKRYFTW MEL, NHL 145 AEPLVGQRW NHL, PRCA, UBC146 SESKTVVTY CRC, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam,OC, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 147 KEVPRSYELHNSCC, NHL, NSCLCadeno, NSCLCsquam, OC, OSCAR, RCC 148 REYNEYENINHL, NSCLCadeno, OSCAR 149 SEKETVAYF MEL, NHL 150 EEVTDRSQLGBC, GC, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC 151 EVDASIFKAWBRCA, CCC, GBC, GBM, HCC, HNSCC, NHL, NSCLCsquam, OC,OSCAR, PACA, PRCA, SCLC, UBC, UEC 152 AELLAKELYCCC, CRC, NHL, NSCLCsquam, UEC 154 AEPGPVITW NHL 155 NEFPVIVRLBRCA, CRC, GBC, GBM, GC, HNSCC, MEL, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 156FEVESLFQKY BRCA, CRC, GBC, GC, GEJC, HCC, HNSCC, MEL, NHL, NSCLCadeno,NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, SCLC, UEC 157 VEIAEAIQLCCC, CRC, HNSCC, NSCLCadeno, NSCLCother, NSCLCsquam, OC 159 GELLGRQSFBRCA, CCC, CRC, MEL, NHL, NSCLCadeno, OC, PACA, RCC, UEC 160 EEETILHFFNHL 161 EEGDTLLHLF CRC, HNSCC, NHL, NSCLCsquam, OSCAR, PACA, UBC, UEC164 SEYSHLTRV RCC 166 NEVLASKY BRCA, CCC, CRC, GBC, GC, HCC,NHL, NSCLCadeno, NSCLCsquam, OC, OSCAR, PRCA, SCLC, UBC, UEC 167KEIGAAVQAL NSCLCadeno, NSCLCother, OC 168 QEIQSLLTNWCCC, CRC, GBM, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam,OSCAR, PACA, RCC, SCLC, UEC 169 EENGEVKEL NHL 171 SEDLAVHLYBRCA, CRC, GC, HCC, HNSCC, NHL, OSCAR, PACA, PRCA, UBC 172 VEDGLFHEFBRCA, CRC, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam,OC, OSCAR, PACA, PRCA, RCC, UBC, UEC 174 TDKSFPNAY PRCA, SCLC 176AENAVSNLSF NHL 177 QENMQIQSF BRCA, CCC, CRC, GC, HCC, HNSCC,MEL, NHL, NSCLCadeno, NSCLCsquam, OC, OSCAR, PACA, SCLC, UBC, UEC 178REYEHYVVTE MEL, NHL, NSCLCadeno, L NSCLCother, OSCAR 179 AEIKQTEEKY HCC180 EEPAFNVSY BRCA, CCC, GBC, NHL, NSCLCadeno, OC, OSCAR 181 GEIKEPLEIBRCA, CCC, CRC, GBC, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OC, OSCAR, PACA, RCC, UEC 182 AQNLSIIQYBRCA, GBC, GC, HCC, MEL, NHL, NSCLCadeno, NSCLCother,NSCLCsquam, OSCAR, PRCA 183 GESQDSTTAL CCC, HNSCC, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OC 184 RMPPFTQAF BRCA, GBC, GC, MEL, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, PACA, PRCA, RCC, SCLC 185SEGDNVESW NHL 187 SDAQRPSSF NSCLCother, RCC 188 YVDAGTPMYGBC, GBM, GC, NSCLCadeno, NSCLCsquam 189 VTEEPQRLFYBRCA, CCC, GBC, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OC, OSCAR, PACA, PRCA, SCLC, UBC, UEC 190HVDQDLTTY GBM, GC, NSCLCadeno, NSCLCother, NSCLCsquam, OC, PACA, PRCA191 ISEAGKDLLY BRCA, CCC, GEJC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam,OSCAR, PACA, PRCA, SCLC, UEC 192 RSDPGGGGL BRCA, CRC, GBC, GBM, GC, AYGEJC, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother,NSCLCsquam, OC, OSCAR, PACA, PRCA, SCLC, UBC, UEC 193 LTDSEKGNSYGBC, GEJC, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam, OC, PRCA 195YSDKEFAGS BRCA, CRC, GBC, GBM, GC, Y GEJC, HCC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OSCAR, PACA,PRCA, RCC, SCLC, UBC, UEC 196 FTDIDGQVY BRCA, CCC, CRC, GBC, GBM,GC, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother,NSCLCsquam, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 197 SLADVHIEVBRCA, CCC, CRC, GBC, GBM, GC, GEJC, HCC, HNSCC, MEL,NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA,PRCA, RCC, SCLC, UBC, UEC 198 KLLGYDVHV BRCA, CRC, NHL, NSCLCother,OC, RCC, UEC 199 AMPDSPAEV HNSCC, NHL, NSCLCadeno 200 VMLQINPKLBRCA, GBC, GBM, HCC, HNSCC, MEL, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OC, OSCAR, RCC, SCLC, UEC 201 ILAAVETRLBRCA, CCC, CRC, GBC, GBM, GC, GEJC, HCC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA,PRCA, RCC, SCLC, UBC, UEC 203 FLLPKVQSI BRCA, HCC, MEL, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR 204 FLLPKVQSIQMEL, NSCLCadeno, NSCLCother, L NSCLCsquam 205 FLINTNSELBRCA, CCC, CRC, GBC, GBM, GC, GEJC, HCC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA,PRCA, RCC, SCLC, UBC, UEC 206 SLMDLQERL BRCA, CCC, CRC, GBC, GBM,GEJC, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother,NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, SCLC, UEC 207 KLSDNILKLCRC, GBC, MEL, NHL, PRCA 208 KLNPQQAPLY MEL, NHL, OSCAR, UEC 209KTLPAMLGTG NHL, OC K 210 RMYSQLKTL MEL, NHL QK 211 ATYNKQPMYCCC, CRC, GBC, GC, MEL, NHL, R NSCLCadeno, NSCLCsquam, OC,OSCAR, RCC, SCLC, UEC 213 RVYNIYIRR NHL, NSCLCadeno, OC 215 KATGAATPKNSCLCsquam 216 RIKAPSRNTIQ NHL K 217 TTVPHVFSKBRCA, CCC, CRC, GBC, GC, HCC, MEL, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, SCLC, UEC 218RVLTGVFTK NHL 220 SISNLVFTY BRCA, CRC, GBC, GC, HCC,HNSCC, MEL, NSCLCadeno, NSCLCsquam, OC, OSCAR, PACA,PRCA, RCC, SCLC, UBC, UEC 221 LLNRHILAH MEL, UEC 222 RYLDEINLLBRCA, CCC, CRC, GBC, GBM, GC, HCC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA,PRCA, RCC, SCLC, UBC, UEC 223 RRMYPPPLI HCC, MEL, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OC, SCLC, UEC 224 VYEYVVERFBRCA, CCC, CRC, GBC, GBM, GC, GEJC, HCC, HNSCC, MEL,NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA,PRCA, RCC, SCLC, UBC, UEC 225 LPARFYQAL BRCA, OSCAR 226 YLNRHLHTWNHL, NSCLCsquam 227 APINKAGSFL MEL, NHL, OC 229 SPLGSLARSSGBC, NHL, OC, OSCAR L 230 KPMKSVLVV BRCA, GC, NHL, NSCLCadeno,OC, OSCAR, UBC 231 MPLSTIREV BRCA, CRC, GBC, GBM, GC,HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam, OC,OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 232 APRPAGSYLBRCA, CCC, CRC, GBC, GBM, GC, HCC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA,PRCA, RCC, SCLC, UBC, UEC 233 SPRVYWLGL NHL 234 SPKESENALNHL, NSCLCsquam 237 SPWLHAPSL GBC, NHL, NSCLCadeno, OC 238 SPRSWIQVQIBRCA, CRC, GBC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother,NSCLCsquam, OC, OSCAR, PACA, RCC, SCLC, UEC 239 APSKTSLIMBRCA, CCC, CRC, GBC, GBM, GC, HCC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA,PRCA, RCC, SCLC, UBC, UEC 240 SPSLPNITL BRCA, CCC, CRC, GBC, GBM, GC,HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother,NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 241 APAPAEKTPVBRCA, CRC, GBC, GBM, GC, HNSCC, MEL, NHL, NSCLCadeno,NSCLCsquam, OC, OSCAR, PACA, PRCA, SCLC, UBC, UEC 242 SPFSFHHVLBRCA, CCC, CRC, GBC, GBM, GC, HCC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA,PRCA, RCC, SCLC, UBC, UEC 243 LPKVQSIQL CCC, HCC, NSCLCadeno,NSCLCsquam, OC, OSCAR, RCC 244 MPSSDTTVTF BRCA, CCC, CRC, GBC, GBM, GC,HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother,NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 245 SPLSHHSQLGC, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR 246 YPGWHSTTICRC, HNSCC, NHL 247 QPSPARAPA GBC, NHL, NSCLCadeno, ELNSCLCsquam, OSCAR, SCLC 248 LPYDSKHQI BRCA, CRC, GBC, GBM, GC,HNSCC, MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC,OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 249 SPADHRGYABRCA, CRC, GBC, HCC, HNSCC, SL MEL, NSCLCadeno, NSCLCother,NSCLCsquam, OC, OSCAR, RCC, SCLC, UBC, UEC 250 VPNLQTVSVBRCA, CCC, CRC, GBC, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno,NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 251 QPRLFTMDLBRCA, CRC, GBC, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OC, OSCAR, PACA, RCC, SCLC, UEC 252 RPHIPISKLBRCA, CRC, GBC, GBM, HCC, HNSCC, MEL, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OC, OSCAR, PACA, RCC, SCLC, UBC, UEC 253RPFADLLGTA BRCA, GBC, GC, HCC, HNSCC, F MEL, NHL, NSCLCadeno,NSCLCsquam, OC, OSCAR, PACA, SCLC, UBC, UEC 254 SPRNLQPQRCRC, GBC, HCC, HNSCC, MEL, AAL NHL, NSCLCadeno, NSCLCsquam,OSCAR, PACA, RCC, SCLC, UBC 255 YPGSDRIML MEL, NHL, NSCLCadeno 256SPYKKLKEAL BRCA, MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam,OSCAR, PRCA, RCC, SCLC, UBC 257 KEFFFVKVF BRCA, CRC, GC, HCC, HNSCC,MEL, NHL, NSCLCadeno, NSCLCsquam, OSCAR, PACA, RCC, SCLC, UBC, UEC 258EELFRDGVN CCC, CRC, HCC, MEL, NHL, W NSCLCadeno, NSCLCsquam, PACA,PRCA, UEC 259 EENTLVQNY BRCA, CRC, HCC, HNSCC, MEL,NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA,PRCA, SCLC, UBC, UEC 260 AEIGEGAYGK BRCA, CCC, CRC, GBM, HCC, VFHNSCC, MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC,OSCAR, PACA, PRCA, UBC, UEC 261 NEIEHIPVW NHL, OSCAR 262 QENQAETHA NHL W263 REAGFQVKA MEL, NHL Y 264 SEDHSGSYW CRC, GC, MEL, NHL, NSCLCadeno,NSCLCsquam 265 QEVDASIFKA BRCA, CCC, CRC, GBC, GC, HCC, WHNSCC, MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC,OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 266 VDASIFKAWBRCA, CRC, GBC, HCC, HNSCC, MEL, NHL, NSCLCadeno,NSCLCsquam, OC, OSCAR, PACA, RCC, SCLC, UBC, UEC 267 KEKFPINGWCRC, HNSCC, NHL, PACA 269 KELEDLNKW BRCA, CRC, GBC, GBM, HCC,HNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, UEC270 AESEDLAVHL MEL, NSCLCsquam 271 AESEDLAVHL CRC, HCC, HNSCC, MEL, NHL,Y NSCLCadeno, NSCLCsquam, OSCAR, PACA 272 KEFELRSSWBRCA, CRC, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno,NSCLCsquam, OSCAR, PACA, PRCA, RCC, UEC 273 AEIEIVKEEFBRCA, CCC, CRC, GBC, GBM, GC, HCC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 274GEAVTDHPD NHL RLW 275 TENPLTKLL BRCA, CRC, HCC, NHL 276 EEEGNLLRSBRCA, CCC, CRC, GBC, GC, W HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother,NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 277 EEGNLLRSWBRCA, CRC, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam,OSCAR, PACA, RCC, UBC, UEC

Example 2

Expression Profiling of Genes Encoding the Peptides of the Invention

Over-presentation or specific presentation of a peptide on tumor cellscompared to normal cells is sufficient for its usefulness inimmunotherapy, and some peptides are tumor-specific despite their sourceprotein occurring also in normal tissues. Still, mRNA expressionprofiling adds an additional level of safety in selection of peptidetargets for immunotherapies. Especially for therapeutic options withhigh safety risks, such as affinity-matured TCRs, the ideal targetpeptide will be derived from a protein that is unique to the tumor andnot found on normal tissues.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated above(see Example 1) after written informed consent had been obtained fromeach patient. Tumor tissue specimens were snap-frozen immediately aftersurgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRI Reagent(Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN,Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from healthy human tissues for RNASeq experiments was obtainedfrom: Asterand (Detroit, Mich., USA & Royston, Herts, UK); Bio-OptionsInc. (Brea, Calif., USA); Geneticist Inc. (Glendale, Calif., USA);ProteoGenex Inc. (Culver City, Calif., USA); Tissue Solutions Ltd(Glasgow, UK). Total RNA from tumor tissues for RNASeq experiments wasobtained from: Asterand (Detroit, Mich., USA & Royston, Herts, UK);BioCat GmbH (Heidelberg, Germany); BioServe (Beltsville, Md., USA);Geneticist Inc. (Glendale, Calif., USA); Istituto Nazionale Tumori“Pascale” (Naples, Italy); ProteoGenex Inc. (Culver City, Calif., USA);University Hospital Heidelberg (Heidelberg, Germany). Quality andquantity of all RNA samples were assessed on an Agilent 2100 Bioanalyzer(Agilent, Waldbronn, Germany) using the RNA 6000 Pico LabChip Kit(Agilent).

RNAseq Eexperiments

Gene expression analysis of—tumor and normal tissue RNA samples wasperformed by next generation sequencing (RNAseq) by CeGaT (Tübingen,Germany). Briefly, sequencing libraries are prepared using the IlluminaHiSeq v4 reagent kit according to the provider's protocol (IlluminaInc., San Diego, Calif., USA), which includes RNA fragmentation, cDNAconversion and addition of sequencing adaptors. Libraries derived frommultiple samples are mixed equimolar and sequenced on the Illumina HiSeq2500 sequencer according to the manufacturer's instructions, generating50 bp single end reads. Processed reads are mapped to the human genome(GRCh38) using the STAR software. Expression data are provided ontranscript level as RPKM (Reads Per Kilobase per Million mapped reads,generated by the software Cufflinks) and on exon level (total reads,generated by the software Bedtools), based on annotations of the ensemblsequence database (Ensembl77). Exon reads are normalized for exon lengthand alignment size to obtain RPKM values.

Exemplary expression profiles of source genes of the present inventionthat are highly over-expressed or exclusively expressed in chroniclymphocytic leukemia, chronic myeloid leukemia and acute myeloidleukemia are shown in FIG. 1. Expression scores for further exemplarygenes are shown in Table 9.

TABLE 9 Expression scores. The table lists peptides fromgenes that are very highly over-expressed intumors compared to a panel of normal tissues(+++), highly over-expressed in tumors comparedto a panel of normal tissues (++) or over-expressed in tumors compared to a panel ofnormal tissues (+). The baseline for this scorewas calculated from measurements of the fol-lowing relevant normal tissues: blood cells,blood vessels, brain, heart, liver, lung,adipose tissue, adrenal gland, bile duct,bladder, bone marrow, cartilage, esophagus,eye, gallbladder, head & neck, kidney, largeintestine, lymph node, nerve, pancreas, para-thyroid, peritoneum, pituitary, pleura, skeletalmuscle, skin, small intestine, spleen, stomach,thyroid gland, trachea, ureter. In case expres-sion data for several samples of the same tissuetype were available, the arithmetic mean of allrespective samples was used for the calculation. Gene  SEQ ID ExpressionNo Sequence AML CLL   1 LTEGHSGNYY +++   2 TMIRIFHRY +++   3YINPAKLTPY +   4 ALDQNKMHY +   5 GTDVLSTRY +   6 VTEGVAQTSFY +   7FMDSESFYY ++   8 STDSAGSSY ++   9 YSHPQYSSY +++  10 YSDIGHLL +  11AAADHHSLY +  12 ATDIVDSQY ++  13 ITDIHIKY +  14 TFDLTVVSY +  15SVADIRNAY +  16 WIGDKSFEY +  17 KAYNRVIFV +  18 YLLPSVVLL +  19SLFEGIYTI +++  20 FSLEDLVRI +  21 FLFDKLLLI +  22 ILHAQTLKI ++  23FAFSGVLRA +  24 KLGPVAVSI +  25 YLNEKSLQL +  26 SLYVQQLKI +  27RLIAKEMNI +  28 VILESIFLK +  29 RIYDEILQSK + +  30 RTYGFVLTF +  31ATFNKLVSY +  32 KTSNIVKIK ++  33 SVFEGDSIVLK ++  34 SVYSETSNMDK +  35ATKSPAKPK ++  36 KAKAAAKPK ++  37 KAKKPAGAAK +  38 KARKSAGAAK +  39IVIQLRAQK +  40 RSKEYIRKK ++  41 SVAHLLSKY +  42 SVSSSTHFTR +  43KLMETSMGF +  44 KVYDPVSEY +  45 VVFPFPVNK ++  46 RVFPSPMRI +  47SVLDLSVHK +  48 RIKPPGPTAVPK +  49 GLLEEALFY +  50 GVFNTLISY +  51ASTTVLALK +  52 KAFNQSSTLTK +++  53 KYIEYYLVL ++  54 QQALNFTRF +  55IFVARLYYF +  56 KYSSGFRNI +  57 RFPPTPPLF +  58 KYLADLPTL +  59GLYEGTGRLF +  60 TQDPHVNAFF +  61 IFKEHNFSF +++  62 YYLSHLERI +  63IYFSNTHFF +  64 SFQSKATVF +++  65 AYLKQVLLF ++  66 SQPAVATSF +  67VFLPSEGFNF + +  68 LYQDRFDYL ++  69 EYNTIKDKF +  70 LYSDIGHLL +  71RYLGKNWSF ++  72 TYVENLRLL +  73 TYPQLEGFKF ++  74 SYADNILSF +  75RFYLLTEHF +  76 KAFSWSSAF +++  77 RPNGNSLFTSA +  78 RPRGLALVL +  79SPVPSHWMVA +  80 KPLFKVSTF +  81 SESPWLHAPSL +  82 APFGFLGMQSL +  83IPVSRPIL ++  84 SPKLQIAAM ++  85 IPVSHPVL +++  86 IPASHPVL +++  87FPAPILRAV +  88 MPDPHLYHQM +  89 FPETVNNLL + +  90 KPKAAKPKA ++  91KPKAAKPKAA ++  92 KAKKPAGAA +  93 KARKSAGAA +  94 KPKAAKPKKAAA +  95KPKAAKPKTA +  96 KPKKAPKSPA +  97 LPFGKIPIL +  98 YPIALTRAEM +  99SPRAINNLVL ++ 100 YPYQERVFL + 101 NPRYPNYMF + 102 LPLSMEAKI + 103IPANTEKASF + 104 RPMTPTQIGPSL ++ 105 NPLTKLLAI ++ + 106 KAFKWFSAL + 107QAAQRTAL + 108 ILAIRQNAL + 109 LGHVRYVL + 110 FGLARIYSF ++ 111 VTLIKYQEL++ 112 APLLRHWEL + 113 DANSRTSQL + 114 HNALRILTF + 115 ELYQRIYAF + 116TLKIRAEVL ++ 117 YIKTAKKL ++ 118 FEKEKKESL + 119 DLRTKEVVF + 120VPPKKHLL + 121 RPKKVNTL + 122 KELPGVKKY ++ 123 EENPGKFLF ++ 124SESLPKEAF + 125 SESTFDRTF + 126 EENKPGIVY + 127 TEYPVFVY + 128GENDRLNHTY + 129 GEGAYGKVF +++ 130 EEEHGKGREY + 131 EEFETIERF + 132GELPAVRDL + 133 AEHNFVAKA ++ 134 SEYADTHYF +++ 135 NEIKVYITF + 136AEYKGRVTL ++ 137 GELGGSVTI ++ 138 SQAPAARAF + 139 RENQVLGSGW ++ 140EYDLKWEF +++ 141 REYEYDLKWEF +++ 142 TEIFKEHNF +++ 143 YEYDLKWEF +++ 144TEGKRYFTW + 145 AEPLVGQRW ++ 146 SESKTVVTY ++ 147 KEVPRSYEL + 148REYNEYENI +++ 149 SEKETVAYF ++ 150 EEVTDRSQL + 151 EVDASIFKAW + 152AELLAKELY ++ 153 KEFEQVPGHL + 154 AEPGPVITW ++ 155 NEFPVIVRL + 156FEVESLFQKY + 157 VEIAEAIQL + 158 GENEDNRIGL ++ 159 GELLGRQSF + 160EEETILHFF + 161 EEGDTLLHLF + + 162 DEAQARAAF ++ 163 EEWMGLLEY ++ 164SEYSHLTRV + 165 VELDLQRSV + 166 NEVLASKY + 167 KEIGAAVQAL + 168QEIQSLLTNW + 169 EENGEVKEL + 170 SENEQRRMF + 171 SEDLAVHLY ++ 172VEDGLFHEF + 173 KEYDFGTQL + 174 TDKSFPNAY + 175 HEIDGKALFL + 176AENAVSNLSF ++ 177 QENMQIQSF + 178 REYEHYWTEL + 179 AEIKQTEEKY + 180EEPAFNVSY + 181 GEIKEPLEI + 182 AQNLSIIQY ++ 183 GESQDSTTAL ++ 184RMPPFTQAF + 185 SEGDNVESW + 186 NEQKIVRF + 187 SDAQRPSSF + 188YVDAGTPMY + 189 VTEEPQRLFY +++ 190 HVDQDLTTY ++ 191 ISEAGKDLLY + + 192RSDPGGGGLAY ++ 193 LTDSEKGNSY ++ 194 YTDKKSIIY + 195 YSDKEFAGSY + 196FTDIDGQVY + 197 SLADVHIEV + 198 KLLGYDVHV + 199 AMPDSPAEV + + 200VMLQINPKL + 201 ILAAVETRL + 202 MVALPMVLV + 203 FLLPKVQSI + 204FLLPKVQSIQL + 205 FLINTNSEL + 206 SLMDLQERL + 207 KLSDNILKL + 208KLNPQQAPLY + 209 KTLPAMLGTGK ++ 210 RMYSQLKTLQK ++ 211 ATYNKQPMYR + 212LLWHWDTTQSLK ++ 213 RVYNIYIRR + 214 ATGAATPKK + 215 KATGAATPK + 216RIKAPSRNTIQK + 217 TTVPHVFSK + 218 RVLTGVFTK + 219 HSYSSPSTK + 220SISNLVFTY + 221 LLNRHILAH + 222 RYLDEINLL + 223 RRMYPPPLI + 224VYEYVVERF + 225 LPARFYQAL + 226 YLNRHLHTW ++ 227 APINKAGSFL + 228SPRITFPSL + 229 SPLGSLARSSL + 230 KPMKSVLVV + 231 MPLSTIREV +++ 232APRPAGSYL + 233 SPRVYWLGL ++ 234 SPKESENAL + 235 SPSLPSRTL + 236RPSNKAPLL + 237 SPWLHAPSL + 238 SPRSWIQVQI +++ 239 APSKTSLIM + 240SPSLPNITL + 241 APAPAEKTPV + 242 SPFSFHHVL + 243 LPKVQSIQL + 244MPSSDTTVTF + 245 SPLSHHSQL + 246 YPGWHSTTI + 247 QPSPARAPAEL + +++ 248LPYDSKHQI + 249 SPADHRGYASL + 250 VPNLQTVSV + 251 QPRLFTMDL + 252RPHIPISKL + + 253 RPFADLLGTAF ++ 254 SPRNLQPQRAAL ++ 255 YPGSDRIML + 256SPYKKLKEAL + 257 KEFFFVKVF + 258 EELFRDGVNW ++ 259 EENTLVQNY + 260AEIGEGAYGKVF +++ 261 NEIEHIPVW + 262 QENQAETHAW + 263 REAGFQVKAY + 264SEDHSGSYW + 265 QEVDASIFKAW + 266 VDASIFKAW + 267 KEKFPINGW + 268NEDKGTKAW + 269 KELEDLNKW + 270 AESEDLAVHL ++ 271 AESEDLAVHLY ++ 272KEFELRSSW + 273 AEIEIVKEEF + 274 GEAVTDHPDRLW ++ 275 TENPLTKLL ++ + 276EEEGNLLRSW ++ 277 EEGNLLRSW ++

Example 3

In Vitro Immunogenicity for MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, the inventors performed investigationsusing an in vitro T-cell priming assay based on repeated stimulations ofCD8+ T cells with artificial antigen presenting cells (aAPCs) loadedwith peptide/MHC complexes and anti-CD28 antibody. This way theinventors could show immunogenicity for HLA-A*02:01, HLA-A*24:02,HLA-A*01:01, HLA-A*03:01, HLA-B*07:02 and HLA-B*44:02 restricted TUMAPsof the invention, demonstrating that these peptides are T-cell epitopesagainst which CD8+ precursor T cells exist in humans (Table 10a andTable 10b).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28antibody, the inventors first isolated CD8+ T cells from fresh HLA-A*02,HLA-A*24, HLA-A*01, HLA-A*03, HLA-B*07 or HLA-B*44 leukapheresisproducts via positive selection using CD8 microbeads (Miltenyi Biotec,Bergisch-Gladbach, Germany) of healthy donors obtained from theUniversity clinics Mannheim, Germany, after informed consent.

PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium(TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,Nürnberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed in a highly defined in vitro system using fourdifferent pMHC molecules per stimulation condition and 8 different pMHCmolecules per readout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung etal., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.6 pm diameter streptavidincoated polystyrene particles (Bangs Laboratories, Illinois, USA).

pMHC used for positive and negative control stimulations wereA*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 280) from modifiedMelan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO.281), respectively.

800.000 beads/200 pl were coated in 96-well plates in the presence of4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 wereadded subsequently in a volume of 200 μl. Stimulations were initiated in96-well plates by co-incubating 1×10⁶ CD8+ T cells with 2×10⁵ washedcoated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell)for 3 days at 37° C. Half of the medium was then exchanged by fresh TCMsupplemented with 80 U/ml IL-2 and incubating was continued for 4 daysat 37° C. This stimulation cycle was performed for a total of threetimes. For the pMHC multimer readout using 8 different pMHC moleculesper condition, a two-dimensional combinatorial coding approach was usedas previously described (Andersen et al., 2012) with minor modificationsencompassing coupling to 5 different fluorochromes. Finally, multimericanalyses were performed by staining the cells with Live/dead near IR dye(Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD,Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BDLSRII SORP cytometer equipped with appropriate lasers and filters wasused. Peptide specific cells were calculated as percentage of total CD8+cells. Evaluation of multimeric analysis was done using the FlowJosoftware (Tree Star, Oreg., USA). In vitro priming of specificmultimer+CD8+ lymphocytes was detected by comparing to negative controlstimulations. Immunogenicity for a given antigen was detected if atleast one evaluable in vitro stimulated well of one healthy donor wasfound to contain a specific CD8+ T-cell line after in vitro stimulation(i.e. this well contained at least 1% of specific multimer+among CD8+T-cells and the percentage of specific multimer+cells was at least 10×the median of the negative control stimulations).

In Vitro Immunogenicity for Chronic Lymphocytic Leukemia, ChronicMyeloid Leukemia and Acute Myeloid Leukemia Peptides

For tested HLA class I peptides, in vitro immunogenicity could bedemonstrated by generation of peptide specific T-cell lines. Exemplaryflow cytometry results after TUMAP-specific multimer staining for 14peptides of the invention are shown in FIGS. 2 to 9 together withcorresponding negative controls. Results for 63 peptides from theinvention are summarized in Table 10a and Table 10b.

TABLE 10a in vitro immunogenicity of HLA class Ipeptides of the invention Exemplary results of in vitro immunogenicityexperiments conducted by the applicant forthe peptides of the invention. <20% = +;20%-49% = ++; 50%-69% = +++; >=70% = ++++ Seq ID Wells positive NoSequence [%] 278 YLDRKLLTL ++++ 279 LYIDRPLPYL ++++

TABLE 10b in vitro immunogenicity of HLA class Ipeptides of the invention Exemplary results of in vitro immunogenicityexperiments conducted by the applicant forthe peptides of the invention. <20% = +;20%-49% = ++; 50%-69% = +++; >=70% = ++++ Seq Wells ID positive NoSequence [%] HLA   1 LTEGHSGNYY “+” A*01  12 ATDIVDSQY “++” A*01  19SLFEGIYTI “++++” A*02  22 ILHAQTLKI “+” A*02  24 KLGPVAVSI “+++” A*02 25 YLNEKSLQL “+” A*02  26 SLYVQQLKI “++” A*02  27 RLIAKEMNI “+” A*02 29 RIYDEILQSK “+” A*03  45 VVFPFPVNK “+” A*03  47 SVLDLSVHK “+” A*03 51 ASTTVLALK “+” A*03  53 KYIEYYLVL “+++” A*24  55 IFVARLYYF “+” A*24 57 RFPPTPPLF “+” A*24  68 LYQDRFDYL “+” A*24  73 TYPQLEGFKF “+” A*24 83 IPVSRPIL “+” B*07  84 SPKLQIAAM “++” B*07  87 FPAPILRAV “+” B*07  89FPETVNNLL “+” B*07  96 KPKKAPKSPA “+” B*07  99 SPRAINNLVL “+” B*07 104RPMTPTQIGPSL “+” B*07 122 KELPGVKKY “++++” B*44 123 EENPGKFLF “++” B*44129 GEGAYGKVF “+++” B*44 133 AEHNFVAKA “+” B*44 134 SEYADTHYF “+++” B*44136 AEYKGRVTL “+” B*44 137 GELGGSVTI “++++” B*44 139 RENQVLGSGW “+++”B*44 140 EYDLKWEF “++++” B*44 142 TEIFKEHNF “++” B*44 143 YEYDLKWEF “+”B*44 145 AEPLVGQRW “++++” B*44 146 SESKTVVTY “+++” B*44 149 SEKETVAYF“+” B*44 154 AEPGPVITW “+++” B*44 158 GENEDNRIGL “++” B*44 162 DEAQARAAF“+” B*44 171 SEDLAVHLY “++++” B*44 176 AENAVSNLSF “++” B*44 182AQNLSIIQY “++++” B*44 192 RSDPGGGGLAY “+” A*01 197 SLADVHIEV “++” A*02207 KLSDNILKL “++” A*02 210 RMYSQLKTLQK “+” A*03 215 KATGAATPK “++” A*03233 SPRVYWLGL “++++” B*07 238 SPRSWIQVQI “+” B*07 247 QPSPARAPAEL “++”B*07 253 RPFADLLGTAF “++++” B*07 254 SPRNLQPQRAAL “+” B*07 258EELFRDGVNW “+” B*44 260 AEIGEGAYGKVF “++” B*44 270 AESEDLAVHL “+” B*44271 AESEDLAVHLY “+” B*44 275 TENPLTKLL “+” B*44 276 EEEGNLLRSW “++” B*44277 EEGNLLRSW “++” B*44

Example 4

Synthesis of Peptides

All peptides were synthesized using standard and well-established solidphase peptide synthesis using the Fmoc-strategy. Identity and purity ofeach individual peptide have been determined by mass spectrometry andanalytical RP-HPLC. The peptides were obtained as white to off-whitelyophilizes (trifluoro acetate salt) in purities of >50%. All TUMAPs arepreferably administered as trifluoro-acetate salts or acetate salts,other salt-forms are also possible.

Example 5

MHC Binding Assays

Candidate peptides for T cell based therapies according to the presentinvention were further tested for their MHC binding capacity (affinity).The individual peptide-MHC complexes were produced by UV-ligandexchange, where a UV-sensitive peptide is cleaved upon UV-irradiation,and exchanged with the peptide of interest as analyzed. Only peptidecandidates that can effectively bind and stabilize the peptide-receptiveMHC molecules prevent dissociation of the MHC complexes. To determinethe yield of the exchange reaction, an ELISA was performed based on thedetection of the light chain (β2m) of stabilized MHC complexes. Theassay was performed as generally described in Rodenko et al. (Rodenko etal., 2006).

96 well MAXISorp plates (NUNC) were coated over night with 2 ug/mlstreptavidin in PBS at room temperature, washed 4× and blocked for1 h at37° C. in 2% BSA containing blocking buffer. RefoldedHLA-A*02:01/MLA-001 monomers served as standards, covering the range of15-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction werediluted 100-fold in blocking buffer. Samples were incubated for 1 h at37° C., washed four times, incubated with 2ug/ml HRP conjugated anti-β2mfor 1h at 37° C., washed again and detected with TMB solution that isstopped with NH₂SO₄. Absorption was measured at 450 nm. Candidatepeptides that show a high exchange yield (preferably higher than 50%,most preferred higher than 75%) are generally preferred for a generationand production of antibodies or fragments thereof, and/or T cellreceptors or fragments thereof, as they show sufficient avidity to theMHC molecules and prevent dissociation of the MHC complexes.

TABLE 11 MHC class I binding scores. Binding ofHLA-class I restricted peptides to HLA-A*01:01 was ranged by peptide exchangeyield: >10% = +; >20% = ++; >50 =+++; >75% = ++++ Seq ID Peptide NoSequence exchange   1 LTEGHSGNYY “++++”   3 YINPAKLTPY “++”   4ALDQNKMHY “+++”   5 GTDVLSTRY “+++”   6 VTEGVAQTSFY “++++”   7 FMDSESFYY“+”   8 STDSAGSSY “++++”   9 YSHPQYSSY “++”  10 YSDIGHLL “++”  11AAADHHSLY “+++”  12 ATDIVDSQY “+++”  13 ITDIHIKY “++++”  14 TFDLTVVSY“++”  15 SVADIRNAY “++”  16 WIGDKSFEY “++” 188 YVDAGTPMY “+++” 189VTEEPQRLFY “++++” 190 HVDQDLTTY “++” 191 ISEAGKDLLY “+++” 192RSDPGGGGLAY “+++” 193 LTDSEKGNSY “+++” 194 YTDKKSIIY “+++” 195YSDKEFAGSY “++++” 196 FTDIDGQVY “+++”

TABLE 12 MHC class I binding scores. Binding ofHLA-class I restricted peptides to HLA-A*02:01 was ranged by peptide exchangeyield: >10% = +; >20% = ++; >50 = +++; >75% = ++++ Seq ID Peptide NoSequence exchange  17 KAYNRVIFV “+++”  18 YLLPSVVLL “++++”  19 SLFEGIYTI“++++”  20 FSLEDLVRI “++”  21 FLFDKLLLI “++++”  22 ILHAQTLKI “+++”  23FAFSGVLRA “++”  24 KLGPVAVSI “++++”  25 YLNEKSLQL “++++”  26 SLYVQQLKI“++++”  27 RLIAKEMNI “++++” 197 SLADVHIEV “++++” 198 KLLGYDVHV “+++” 199AMPDSPAEV “++++” 200 VMLQINPKL “+++” 201 ILAAVETRL “+++” 203 FLLPKVQSI“++++” 204 FLLPKVQSIQL “+++” 205 FLINTNSEL “++++” 206 SLMDLQERL “+++”207 KLSDNILKL “++++”

TABLE 13 MHC class I binding scores. Binding ofHLA-class I restricted peptides to HLA-A*03:01 was ranged by peptide exchangeyield: >10% = +; >20% = ++; >50 = +++; >75% = ++++ Seq ID Peptide NoSequence exchange  28 VILESIFLK “++”  29 RIYDEILQSK “++”  30 RTYGFVLTF“++”  32 KTSNIVKIK “++”  33 SVFEGDSIVLK “++”  34 SVYSETSNMDK “+++”  35ATKSPAKPK “+++”  36 KAKAAAKPK “++”  37 KAKKPAGAAK “+++”  38 KARKSAGAAK“+++”  40 RSKEYIRKK “+”  41 SVAHLLSKY “++”  42 SVSSSTHFTR “++”  43KLMETSMGF “++”  44 KVYDPVSEY “++”  45 VVFPFPVNK “+++”  46 RVFPSPMRI “++” 47 SVLDLSVHK “++”  48 RIKPPGPTAVPK “+++”  49 GLLEEALFY “++”  51ASTTVLALK “++”  52 KAFNQSSTLTK “+++” 208 KLNPQQAPLY “++” 209 KTLPAMLGTGK“++” 210 RMYSQLKTLQK “+++” 211 ATYNKQPMYR “+++” 212 LLWHWDTTQSLK “+” 213RVYNIYIRR “++” 214 ATGAATPKK “+++” 215 KATGAATPK “++” 216 RIKAPSRNTIQK“++” 217 TTVPHVFSK “++” 218 RVLTGVFTK “++” 219 HSYSSPSTK “+++” 220SISNLVFTY “++” 221 LLNRHILAH “+++”

TABLE 14 MHC class I binding scores. Binding ofHLA-class I restricted peptides to HLA-A*24:02 was ranged by peptide exchangeyield: >10% = +; >20% = ++; >50 = +++; >75% = ++++ Seq ID Peptide NoSequence exchange  53 KYIEYYLVL “++++”  55 IFVARLYYF “++++”  56KYSSGFRNI “+++”  57 RFPPTPPLF “++++”  58 KYLADLPTL “++++”  61 IFKEHNFSF“++++”  62 YYLSHLERI “++++”  63 IYFSNTHFF “+++”  64 SFQSKATVF “++”  67VFLPSEGFNF “+++”  68 LYQDRFDYL “++++”  69 EYNTIKDKF “+++”  70 LYSDIGHLL“+++”  71 RYLGKNWSF “++++”  72 TYVENLRLL “+++”  73 TYPQLEGFKF “++++”  74SYADNILSF “++++”  75 RFYLLTEHF “+++” 222 RYLDEINLL “+++”

TABLE 15 MHC class I binding scores. Binding ofHLA-class I restricted peptides to HLA-B*07:02 was ranged by peptide exchangeyield: >10% = +; >20% = ++; >50 = +++; >75% = ++++ Seq ID Peptide NoSequence exchange  77 RPNGNSLFTSA “+++”  78 RPRGLALVL “+++”  79SPVPSHWMVA “+++”  80 KPLFKVSTF “++”  81 SESPWLHAPSL “+++”  82APFGFLGMQSL “+++”  83 IPVSRPIL “+++”  84 SPKLQIAAM “+++”  85 IPVSHPVL“++”  86 IPASHPVL “++”  87 FPAPILRAV “+++”  88 MPDPHLYHQM “++”  89FPETVNNLL “++”  90 KPKAAKPKA “++”  91 KPKAAKPKAA “++”  92 KAKKPAGAA “++” 93 KARKSAGAA “+++”  94 KPKAAKPKKAAA “++”  95 KPKAAKPKTA “++”  96KPKKAPKSPA “+++”  97 LPFGKIPIL “++”  98 YPIALTRAEM “+++”  99 SPRAINNLVL“++++” 100 YPYQERVFL “++” 101 NPRYPNYMF “+++” 102 LPLSMEAKI “++” 103IPANTEKASF “++” 104 RPMTPTQIGPSL “+++” 105 NPLTKLLAI “+++” 106 KAFKWFSAL“++” 225 LPARFYQAL “++++” 226 YLNRHLHTW “++” 227 APINKAGSFL “++++” 228SPRITFPSL “++++” 229 SPLGSLARSSL “++++” 230 KPMKSVLVV “+++” 231MPLSTIREV “++” 232 APRPAGSYL “+++” 233 SPRVYWLGL “+++” 234 SPKESENAL“++” 235 SPSLPSRTL “++” 236 RPSNKAPLL “+++” 237 SPWLHAPSL “++” 238SPRSWIQVQI “+++” 239 APSKTSLIM “++” 240 SPSLPNITL “+++” 241 APAPAEKTPV“+++” 242 SPFSFHHVL “++” 243 LPKVQSIQL “++” 244 MPSSDTTVTF “+++” 245SPLSHHSQL “++” 246 YPGWHSTTI “++” 247 QPSPARAPAEL “++” 248 LPYDSKHQI“++” 249 SPADHRGYASL “++” 250 VPNLQTVSV “++” 251 QPRLFTMDL “+++” 252RPHIPISKL “++” 253 RPFADLLGTAF “+++” 254 SPRNLQPQRAAL “+++” 255YPGSDRIML “++”

TABLE 16 MHC class I binding scores. Binding ofHLA-class I restricted peptides to HLA-B*44:02 was ranged by peptide exchangeyield: >10% = +; >20% = ++; >50 = +++; >75% = ++++ Seq ID Peptide NoSequence exchange 122 KELPGVKKY “++” 123 EENPGKFLF “+++” 124 SESLPKEAF“++” 125 SESTFDRTF “+++” 126 EENKPGIVY “++” 127 TEYPVFVY “+” 128GENDRLNHTY “++” 129 GEGAYGKVF “++++” 130 EEEHGKGREY “++” 131 EEFETIERF“++” 132 GELPAVRDL “++” 133 AEHNFVAKA “+++” 134 SEYADTHYF “+++” 135NEIKVYITF “+++” 136 AEYKGRVTL “++++” 137 GELGGSVTI “+++” 138 SQAPAARAF“++” 139 RENQVLGSGW “+++” 140 EYDLKWEF “++” 141 REYEYDLKWEF “+++” 142TEIFKEHNF “++” 143 YEYDLKWEF “+++” 144 TEGKRYFTW “+++” 145 AEPLVGQRW“+++” 146 SESKTVVTY “+++” 147 KEVPRSYEL “++” 148 REYNEYENI “++” 149SEKETVAYF “+++” 150 EEVTDRSQL “++” 151 EVDASIFKAW “++” 152 AELLAKELY“++” 153 KEFEQVPGHL “++” 154 AEPGPVITW “+++” 155 NEFPVIVRL “+++” 156FEVESLFQKY “+++” 157 VEIAEAIQL “+++” 158 GENEDNRIGL “++” 159 GELLGRQSF“+++” 160 EEETILHFF “++” 161 EEGDTLLHLF “+++” 162 DEAQARAAF “++” 163EEWMGLLEY “++++” 164 SEYSHLTRV “++” 165 VELDLQRSV “++” 166 NEVLASKY “+”167 KEIGAAVQAL “+++” 168 QEIQSLLTNW “+++” 169 EENGEVKEL “++” 170SENEQRRMF “+++” 171 SEDLAVHLY “++” 172 VEDGLFHEF “+++” 173 KEYDFGTQL“++” 174 TDKSFPNAY “+” 175 HEIDGKALFL “++” 176 AENAVSNLSF “++” 177QENMQIQSF “+++” 178 REYEHYWTEL “+++” 179 AEIKQTEEKY “++” 180 EEPAFNVSY“++” 181 GEIKEPLEI “++” 182 AQNLSIIQY “++” 183 GESQDSTTAL “++” 184RMPPFTQAF “++” 185 SEGDNVESW “+++” 186 NEQKIVRF “+” 187 SDAQRPSSF “+”257 KEFFFVKVF “+++” 258 EELFRDGVNW “+++” 259 EENTLVQNY “++” 260AEIGEGAYGKVF “+++” 261 NEIEHIPVW “+++” 262 QENQAETHAW “+++” 263REAGFQVKAY “+++” 264 SEDHSGSYW “+++” 265 QEVDASIFKAW “+++” 266 VDASIFKAW“++” 267 KEKFPINGW “+++” 268 NEDKGTKAW “++” 269 KELEDLNKW “+++” 270AESEDLAVHL “++” 271 AESEDLAVHLY “++” 272 KEFELRSSW “+++” 273 AEIEIVKEEF“++” 274 GEAVTDHPDRLW “+++” 275 TENPLTKLL “++++” 276 EEEGNLLRSW “+++”277 EEGNLLRSW “+++”

Example 6

Peptide-MHC Class I Stability

The peptide-MHC stability for HLA-B*08:01 peptides was performed byImmunAware (Copenhagen, Denmark). The data were obtained using aproximity based, homogenous, real-time assay to measure the dissociationof peptides from HLA class I molecules. First human recombinantHLA-B*08:01 and b2m were expressed in E. coli and purified in a seriesof liquid chromatography based steps (Ferre et al., 2003; Ostergaard etal., 2001). Afterwards, the stability of a peptide-MHC complex (pMHC)can be determined by measuring the amount of b2m associated with the MHCheavy chain over time at 37° C. (Harndahl et al., 2012). The stabilityof each pMHC, expressed as the half life of b2m associated with therespective heavy chain, was calculated by fitting the data to aone-phase dissociation equation.

The pMHC stability were measured in three independent experiments andthe peptides in question, for HLA-B*08:01, were found to span the rangefrom weak-binders (+) to very stable binders (++++). The mean half-life(T1/2) is shown in Table 17.

TABLE 17 Mean half-life (T1/2) based on threeindividual measurements. T1/2 > 2 h = +;T1/2 > 4 h  = ++; T1/2 > 6 h  = +++; T1/2>/ 10 h  = ++++ Seq IDMean Half-life No Sequence (T1/2) 107 QAAQRTAL ++ 108 ILAIRQNAL ++ 109LGHVRYVL + 110 FGLARIYSF + 112 APLLRHWEL + 113 DANSRTSQL +++ 114HNALRILTF ++ 115 ELYQRIYAF ++ 116 TLKIRAEVL +++ 117 YIKTAKKL ++ 118FEKEKKESL +++ 119 DLRTKEVVF ++ 120 VPPKKHLL + 121 RPKKVNTL +

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1. A method of treating a patient who has cancer, comprisingadministering to said patient a population of activated T cells thatkill cancer cells that present a peptide consisting of the amino acidsequence of SEQ ID NO: 53, wherein said cancer is selected from thegroup consisting of chronic lymphocytic leukemia, gastric cancer,non-small cell lung cancer adenocarcinoma, squamous cell non-small celllung cancer, and uterine cancer.
 2. The method of claim 1, wherein the Tcells are autologous to the patient.
 3. The method of claim 1, whereinthe T cells are obtained from a healthy donor.
 4. The method of claim 1,wherein the T cells are obtained from tumor infiltrating lymphocytes orperipheral blood mononuclear cells.
 5. The method of claim 1, whereinthe activated T cells are expanded in vitro.
 6. The method of claim 1,wherein the population of activated T cells are administered in the formof a composition.
 7. The method of claim 6, wherein the compositionfurther comprises an adjuvant.
 8. The method of claim 7, wherein theadjuvant is selected from anti-CD40 antibody, imiquimod, resiquimod,GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon-alpha,interferon-beta, CpG oligonucleotides and derivatives, poly-(I:C) andderivatives, RNA, sildenafil, particulate formulations with poly(lactideco-glycolide) (PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7,IL-12, IL-13, IL-15, IL-21, and IL-23.
 9. The method of claim 1, whereinthe activated T cells are cytotoxic T cells produced by contacting Tcells with an antigen presenting cell that expresses the peptide in acomplex with an WIC class I molecule on the surface of the antigenpresenting cell, for a period of time sufficient to activate said Tcell.
 10. The method of claim 9, wherein the antigen presenting cell isinfected with a recombinant virus expressing the peptide.
 11. The methodof claim 1, wherein the cancer is chronic lymphocytic leukemia.
 12. Themethod of claim 1, wherein the cancer is gastric cancer.
 13. The methodof claim 1, wherein the cancer is uterine cancer.
 14. The method ofclaim 1, wherein the cancer is non-small cell lung canceradenocarcinoma.
 15. A method of eliciting an immune response in apatient who has cancer, comprising administering to said patient apopulation of activated T cells that kill cancer cells that present apeptide consisting of the amino acid sequence of SEQ ID NO: 53, whereinsaid cancer is selected from the group consisting of chronic lymphocyticleukemia, gastric cancer, non-small cell lung cancer adenocarcinoma,squamous cell non-small cell lung cancer, and uterine cancer.
 16. Themethod of claim 15, wherein the activated T cells are cytotoxic T cellsproduced by contacting T cells with an antigen presenting cell thatexpresses the peptide in a complex with an WIC class I molecule on thesurface of the antigen presenting cell, for a period of time sufficientto activate said T cell.
 17. The method of claim 15, wherein the canceris chronic lymphocytic leukemia.
 18. The method of claim 15, wherein thecancer is gastric cancer.
 19. The method of claim 15, wherein the canceris uterine cancer.
 20. The method of claim 15, wherein the cancer isnon-small cell lung cancer adenocarcinoma.